Methods for operating an array of chemically-sensitive sensors

ABSTRACT

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/691,923 filed 22 Jan. 2010, which is a continuation of U.S. patentapplication Ser. No. 12/002,291, filed 14 Dec. 2007, which claimspriority to U.S. Provisional Application No. 60/956,324, filed 16 Aug.2007, U.S. Provisional Application No. 60/948,748, filed 10 Jul. 2007,and U.S. Provisional Application No. 60/870,073, filed 14 Dec. 2006; theentire contents of each of which are incorporated herein by reference.

REFERENCE TO BIOLOGICAL SEQUENCE DISCLOSURE

This application contains nucleotide sequence and/or amino acid sequencedisclosure in computer readable form and a written sequence listing, theentire contents of both of which are expressly incorporated by referencein their entirety as though fully set forth herein.

FIELD OF THE DISCLOSURE

The present disclosure is directed generally to inventive methods andapparatus relating to detection and measurement of one or more analytesvia electronic sensors.

BACKGROUND

Electronic devices and components have found numerous applications inchemistry and biology (more generally, “life sciences”), especially fordetection and measurement of various aspects of chemical reactions andsubstance composition. One such electronic device is referred to as anion-sensitive field effect transistor, often denoted in the relevantliterature as ISFET (or pHFET). ISFETs conventionally have beenexplored, primarily in the academic and research community, tofacilitate measurement of the hydrogen ion concentration of a solution(commonly denoted as “pH”).

More specifically, an ISFET is an impedance transformation device thatoperates in a manner similar to that of a MOSFET (Metal OxideSemiconductor Field Effect Transistor), and is particularly configuredto selectively measure ion activity in a solution (e.g., hydrogen ionsin the solution are the “analyte”). A detailed theory of operation of anISFET is given in “Thirty years of ISFETOLOGY: what happened in the past30 years and what may happen in the next 30 years,” P. Bergveld, Sens.Actuators, 88 (2003), pp. 1-20, which publication is hereby incorporatedherein by reference.

FIG. 1 illustrates a cross-section of a p-type (p-channel) ISFET 50fabricated using a conventional CMOS (Complimentary Metal OxideSemiconductor) process. P-type ISFET fabrication is based on a p-typesilicon substrate 52, in which an n-type well 54 forming a transistor“body” is formed. Highly doped p-type (p+) regions S and D, constitutinga source 56 and a drain 58 of the ISFET, are formed within the n-typewell 54. A highly doped n-type (n+) region B is also formed within then-type well to provide a conductive body (or “bulk”) connection 62 tothe n-type well. An oxide layer 65 is disposed above the source, drainand body connection regions, through which openings are made to provideelectrical connections (via electrical conductors) to these regions; forexample, metal contact 66 serves as a conductor to provide an electricalconnection to the drain 58, and metal contact 68 serves as a conductorto provide a common connection to the source 56 and n-type well 54, viathe highly conductive body connection 62. A polysilicon gate 64 isformed above the oxide layer at a location above a region 60 of then-type well 54, between the source 56 and the drain 58. Because it isdisposed between the polysilicon gate 64 and the transistor body (i.e.,the n-type well), the oxide layer 65 often is referred to as the “gateoxide.”

Like a MOSFET, the operation of an ISFET is based on the modulation ofcharge concentration caused by a MOS (Metal-Oxide-Semiconductor)capacitance constituted by the polysilicon gate 64, the gate oxide 65and the region 60 of the n-type well 54 between the source and thedrain. When a negative voltage is applied across the gate and sourceregions (VGs<0 Volts), a “p-channel” 63 is created at the interface ofthe region 60 and the gate oxide 65 by depleting this area of electrons.This p-channel 63 extends between the source and the drain, and electriccurrent is conducted through the p-channel when the gate-sourcepotential V_(GS) is negative enough to attract holes from the sourceinto the channel. The gate-source potential at which the channel 63begins to conduct current is referred to as the transistor's thresholdvoltage V_(TH) (the transistor conducts when V_(GS) has an absolutevalue greater than the threshold voltage V_(TH)). The source is so namedbecause it is the source of the charge carriers (holes for a p-channel)that flow through the channel 63; similarly, the drain is where thecharge carriers leave the channel 63.

In the ISFET 50 of FIG. 1, the n-type well 54 (transistor body), via thebody connection 62, is forced to be biased at a same potential as thesource 56 (i.e., V_(SB)=0 Volts), as seen by the metal contact 68connected to both the source 56 and the body connection 62. Thisconnection prevents forward biasing of the p+ source region and then-type well, and thereby facilitates confinement of charge carriers tothe area of the region 60 in which the channel 63 may be formed. Anypotential difference between the source 56 and the body/n-type well 54(a non-zero source-to-body voltage V_(SB)) affects the threshold voltageV_(TH) of the ISFET according to a nonlinear relationship, and iscommonly referred to as the “body effect,” which in many applications isundesirable.

As also shown in FIG. 1, the polysilicon gate 64 of the ISFET 50 iscoupled to multiple metal layers disposed within one or more additionaloxide layers 75 disposed above the gate oxide 65 to form a “floatinggate” structure 70. The floating gate structure is so named because itis electrically isolated from other conductors associated with theISFET; namely, it is sandwiched between the gate oxide 65 and apassivation layer 72. In the ISFET 50, the passivation layer 72constitutes an ion-sensitive membrane that gives rise to theion-sensitivity of the device; i.e., the presence of ions in an “analytesolution” 74 (a solution containing ions of interest) in contact withthe passivation layer 72, particularly in a sensitive area 78 above thefloating gate structure 70, alters the electrical characteristics of theISFET so as to modulate a current flowing through the p-channel 63between the source 56 and the drain 58. The passivation layer 72 maycomprise any one of a variety of different materials to facilitatesensitivity to particular ions; for example, passivation layerscomprising silicon nitride or silicon oxynitride generally providesensitivity to hydrogen ion concentration (pH) in the analyte solution74, whereas passivation layers comprising polyvinyl chloride containingvalinomycin provide sensitivity to potassium ion concentration in theanalyte solution (materials suitable for passivation layers andsensitive to other ions such as sodium, silver, iron, bromine, iodine,calcium, and nitrate, for example, are known).

With respect to ion sensitivity, an electric potential difference,commonly referred to as a “surface potential,” arises at thesolid/liquid interface of the passivation layer 72 and the analytesolution 74 as a function of the ion concentration in the sensitive area78 due to a chemical reaction (e.g., usually involving the dissociationof oxide surface groups by the ions in the analyte solution in proximityto the sensitive area 78). This surface potential in turn affects thethreshold voltage V_(TH) of the ISFET; thus, it is the threshold voltageV_(TH) of the ISFET that varies with changes in ion concentration in theanalyte solution 74 in proximity to the sensitive area 78.

FIG. 2 illustrates an electric circuit representation of the p-channelISFET 50 shown in FIG. 1. With reference again to FIG. 1, a referenceelectrode 76 (a conventional Ag/AgCl electrode) in the analyte solution74 determines the electric potential of the bulk of the analyte solutionitself and is analogous to the gate terminal of a conventional MOSFET,as shown in FIG. 2. In a linear or non-saturated operating region of theISFET, the drain current I_(D) is given as:

$\begin{matrix}{{I_{D} = {{\beta\left( {V_{GS} - V_{TH} - {\frac{1}{2}V_{DS}}} \right)} \cdot V_{DS}}},} & (1)\end{matrix}$

where V_(DS) is the voltage between the drain and the source, and β is atransconductance parameter (in units of Amps/Volts²) given by:

$\begin{matrix}{{\beta = {\mu\;{C_{ox}\left( \frac{W}{L} \right)}}},} & (2)\end{matrix}$

where μ represents the carrier mobility, C_(ox) is the gate oxidecapacitance per unit area, and the ratio W/L is the width to lengthratio of the channel 63. If the reference electrode 76 provides anelectrical reference or ground (V_(G)=0 Volts), and the drain currentI_(D) and the drain-to-source voltage V_(DS) are kept constant,variations of the source voltage V_(S) of the ISFET directly trackvariations of the threshold voltage V_(TH), according to Eq. (1); thismay be observed by rearranging Eq. (1) as:

$\begin{matrix}{V_{S} = {{- V_{TH}} - {\left( {\frac{I_{D}}{\beta\; V_{DS}} + \frac{V_{DS}}{2}} \right).}}} & (3)\end{matrix}$

Since the threshold voltage V_(TH) of the ISFET is sensitive to ionconcentration as discussed above, according to Eq. (3) the sourcevoltage V_(S) provides a signal that is directly related to the ionconcentration in the analyte solution 74 in proximity to the sensitivearea 78 of the ISFET. In exemplary conventional ISFETs employing asilicon nitride or silicon oxynitride passivation layer 72 forpH-sensitivity, a threshold voltage sensitivities ΔV_(TH) (i.e., achange in threshold voltage with change in pH of the analyte solution)of approximately 30 mV/pH to 50 mV/pH have been observed (with atheoretical maximum sensitivity of 59.2 mV/pH at 298 degrees Kelvin).

Prior research efforts to fabricate ISFETs for pH measurements based onconventional CMOS processing techniques typically have aimed to achievehigh signal linearity over a pH range from 1-14. Using an exemplarythreshold sensitivity of approximately 50 mV/pH, and considering Eq. (3)above, this requires a linear operating range of approximately 700 mVfor the source voltage V_(S). As discussed above in connection with FIG.1, the threshold voltage V_(TH) of ISFETs (as well as MOSFETs) isaffected by any voltage V_(SB) between the source and the body (n-typewell 54). More specifically, the threshold voltage V_(TH) is a nonlinearfunction of a nonzero source-to-body voltage V_(SB). Accordingly, so asto avoid compromising linearity due to a difference between the sourceand body voltage potentials (i.e., to mitigate the “body effect”), asshown in FIG. 1 the source 56 and body connection 62 of the ISFET 50often are coupled to a common potential via the metal contact 68. Thisbody-source coupling also is shown in the electric circuitrepresentation of the ISFET 50 shown in FIG. 2.

Previous efforts to fabricate two-dimensional arrays of ISFETs based onthe ISFET design of FIG. 1 have resulted in a maximum of 256 ISFETsensor elements, or “pixels,” in an array (i.e., a 16 pixel by 16 pixelarray). Exemplary research in ISFET array fabrication are reported inthe publications “A large transistor-based sensor array chip for directextracellular imaging,” M. J. Milgrew, M. O. Riehle, and D. R. S.Cumming, Sensors and Actuators, B: Chemical, 111-112, (2005), pp.347-353, and “The development of scalable sensor arrays using standardCMOS technology,” M. J. Milgrew, P. A. Hammond, and D. R. S. Cumming,Sensors and Actuators, B: Chemical, 103, (2004), pp. 37-42, whichpublications are incorporated herein by reference and collectivelyreferred to hereafter as “Milgrew et al.” Other research effortsrelating to the realization of ISFET arrays are reported in thepublications “A very large integrated pH-ISFET sensor array chipcompatible with standard CMOS processes,” T. C. W. Yeow, M. R. Haskard,D. E. Mulcahy, H. I. Seo and D. H. Kwon, Sensors and Actuators B:Chemical, 44, (1997), pp. 434-440 and “Fabrication of a two-dimensionalpH image sensor using a charge transfer technique,” Hizawa, T., Sawada,K., Takao, H., Ishida, M., Sensors and Actuators, B: Chemical 117 (2),2006, pp. 509-515, which publications also are incorporated herein byreference.

FIG. 3 illustrates one column 85 _(j) of a two-dimensional ISFET arrayaccording to the design of Milgrew et al. The column 85 _(j) includessixteen (16) pixels 80 ₁ through 80 ₁₆ and, as discussed further belowin connection with FIG. 7, a complete two-dimensional array includessixteen (16) such columns 85 _(j) (j=1, 2, 3, . . . 16) arranged side byside. As shown in FIG. 3, a given column 85 _(j) includes a currentsource I_(SOURCEj) that is shared by all pixels of the column, and ISFETbias/readout circuitry 82 _(j) (including current sink I_(SINKj)) thatis also shared by all pixels of the column. Each ISFET pixel 80 ₁through 80 ₁₆ includes a p-channel ISFET 50 having an electricallycoupled source and body (as shown in FIGS. 1 and 2), plus two switchesS1 and S2 that are responsive to one of sixteen row select signals(RSEL₁ through RSEL₁₆, and their complements). As discussed below inconnection with FIG. 7, a row select signal and its complement aregenerated simultaneously to “enable” or select a given pixel of thecolumn 85 _(j), and such signal pairs are generated in some sequence tosuccessively enable different pixels of the column one at a time.

As shown in FIG. 3, the switch S2 of each pixel 80 in the design ofMilgrew et al. is implemented as a conventional n-channel MOSFET thatcouples the current source I_(SOURCEj) to the source of the ISFET 50upon receipt of the corresponding row select signal. The switch S1 ofeach pixel 80 is implemented as a transmission gate, i.e., a CMOS pairincluding an n-channel MOSFET and a p-channel MOSFET, that couples thesource of the ISFET 50 to the bias/readout circuitry 82 j upon receiptof the corresponding row select signal and its complement. An example ofthe switch S1 ₁ of the pixel 80 ₁ is shown in FIG. 4, in which thep-channel MOSFET of the transmission gate is indicated as S1 _(1P) andthe n-channel MOSFET is indicated as S1 _(IN). In the design of Milgrewet al., a transmission gate is employed for the switch S1 of each pixelso that, for an enabled pixel, any ISFET source voltage within the powersupply range V_(DD) to V_(SS) may be applied to the bias/readoutcircuitry 82 _(j) and output by the column as the signal V_(Sj). Fromthe foregoing, it should be appreciated that each pixel 80 in the ISFETsensor array design of Milgrew et al. includes four transistors, i.e., ap-channel ISFET, a CMOS-pair transmission gate including an n-channelMOSFET and a p-channel MOSFET for switch S1, and an n-channel MOSFET forswitch S2.

As also shown in FIG. 3, the bias/readout circuitry 82 _(j) employs asource-drain follower configuration in the form of a Kelvin bridge tomaintain a constant drain-source voltage V_(DSj) and isolate themeasurement of the source voltage V_(DSj) from the constant draincurrent I_(SOURCEj) for the ISFET of an enabled pixel in the column 85_(j). To this end, the bias/readout circuitry 82 _(j) includes twooperational amplifiers A1 and A2, a current sink I_(SINKj), and aresistor R_(SDj). The voltage developed across the resistor R_(SDJ) dueto the current I_(SINKj) flowing through the resistor is forced by theoperational amplifiers to appear across the drain and source of theISFET of an enabled pixel as a constant drain-source voltage V_(DSJ).Thus, with reference again to Eq. (3), due to the constant V_(DSJ) andthe constant I_(SOURCEj), the source voltage V_(Sj) of the ISFET of theenabled pixel provides a signal corresponding to the ISFETs thresholdvoltage V_(TH), and hence a measurement of pH in proximity to the ISFETssensitive area (see FIG. 1). The wide dynamic range for the sourcevoltage V_(Sj) provided by the transmission gate S1 ensures that a fullrange of values from 1-14 may be measured, and the source-bodyconnection of each ISFET ensures sufficient linearity of the ISFETsthreshold voltage over the full pH measurement range.

In the column design of Milgrew et al. shown in FIG. 3, it should beappreciated that for the Kelvin bridge configuration of the columnbias/readout circuitry 82 _(j) to function properly, a p-channel ISFET50 as shown in FIG. 1 must be employed in each pixel; more specifically,an alternative implementation based on the Kelvin bridge configurationis not possible using an n-channel ISFET. With reference again to FIG.1, for an n-channel ISFET based on a conventional CMOS process, then-type well 54 would not be required, and highly doped n-type regionsfor the drain and source would be formed directly in the p-type siliconsubstrate 52 (which would constitute the transistor body). For n-channelFET devices, the transistor body typically is coupled to electricalground. Given the requirement that the source and body of an ISFET inthe design of Milgrew et al. are electrically coupled together tomitigate nonlinear performance due to the body effect, this would resultin the source of an n-channel ISFET also being connected to electricalground (i.e., V_(S)=V_(B)=0 Volts), thereby precluding any useful outputsignal from an enabled pixel. Accordingly, the column design of Milgrewet al. shown in FIG. 3 requires p-channel ISFETs for proper operation.

It should also be appreciated that in the column design of Milgrew etal. shown in FIG. 3, the two n-channel MOSFETs required to implement theswitches S1 and S2 in each pixel cannot be formed in the n-type well 54shown in FIG. 1, in which the p-channel ISFET for the pixel is formed;rather, the n-channel MOSFETs are formed directly in the p-type siliconsubstrate 52, beyond the confines of the n-type well 54 for the ISFET.FIG. 5 is a diagram similar to FIG. 1, illustrating a widercross-section of a portion of the p-type silicon substrate 52corresponding to one pixel 80 of the column 85 _(j) shown in FIG. 3, inwhich the n-type well 54 containing the drain 58, source 56 and bodyconnection 62 of the ISFET 50 is shown alongside a first n-channelMOSFET corresponding to the switch S2 and a second n-channel MOSFET S1_(1N) constituting one of the two transistors of the transmission gateS1 ₁ shown in FIG. 4.

Furthermore, in the design of Milgrew et al., the p-channel MOSFETrequired to implement the transmission gate S1 in each pixel (e.g., seeS1 _(1p) in FIG. 4) cannot be formed in the same n-type well in whichthe p-channel ISFET 50 for the pixel is formed. IA particular, becausethe body and source of the p-channel ISFET are electrically coupledtogether, implementing the p-channel MOSFET S1 _(1p) in the same n-wellas the p-channel ISFET 50 would lead to unpredictable operation of thetransmission gate, or preclude operation entirely. Accordingly, twoseparate n-type wells are required to implement each pixel in the designof Milgrew et al. FIG. 6 is a diagram similar to FIG. 5, showing across-section of another portion of the p-type silicon substrate 52corresponding to one pixel 80, in which the n-type well 54 correspondingto the ISFET 50 is shown alongside a second n-type well 55 in which isformed the p-channel MOSFET S1 _(1P) constituting one of the twotransistors of the transmission gate S1 ₁ shown in FIG. 4. It should beappreciated that the drawings in FIGS. 5 and 6 are not to scale and maynot exactly represent the actual layout of a particular pixel in thedesign of Milgrew et al.; rather these figures are conceptual in natureand are provided primarily to illustrate the requirements of multiplen-wells, and separate n-channel MOSFETs fabricated outside of then-wells, in the design of Milgrew et al.

The array design of Milgrew et al. was implemented using a 0.35micrometer (μm) conventional CMOS fabrication process. In this process,various design rules dictate minimum separation distances betweenfeatures. For example, according to the 0.35 μm CMOS design rules, withreference to FIG. 6, a distance “a” between neighboring n-wells must beat least three (3) micrometers. A distance “a/2” also is indicated inFIG. 6 to the left of the n-well 54 and to the right of the n-well 55 toindicate the minimum distance required to separate the pixel 80 shown inFIG. 6 from neighboring pixels in other columns to the left and right,respectively. Additionally, according to the 0.35 μm CMOS design rules,a distance “b” shown in FIG. 6 representing the width in cross-sectionof the n-type well 54 and a distance “c” representing the width incross-section of the n-type well 55 are each on the order ofapproximately 3 μm to 4 μm (within the n-type well, an allowance of 1.2μm is made between the edge of the n-well and each of the source anddrain, and the source and drain themselves have a width on the order of0.7 μm). Accordingly, a total distance “d” shown in FIG. 6 representingthe width of the pixel 80 in cross-section is on the order ofapproximately 12 μm to 14 μm. In one implementation, Milgrew et al.report an array based on the column/pixel design shown in FIG. 3comprising geometrically square pixels each having a dimension of 12.8μm by 12.8 μm.

In sum, the ISFET pixel design of Milgrew et al. is aimed at ensuringaccurate hydrogen ion concentration measurements over a pH range of1-14. To ensure measurement linearity, the source and body of eachpixel's ISFET are electrically coupled together. To ensure a full rangeof pH measurements, a transmission gate S1 is employed in each pixel totransmit the source voltage of an enabled pixel. Thus, each pixel of<Milgrew's array requires four transistors (p-channel ISFET, p-channelMOSFET, and two n-channel MOSFETs) and two separate n-wells (FIG. 6).Based on a 0.35 micrometer conventional CMOS fabrication process and thecorresponding design rules, the pixels of such an array have a minimumsize appreciably greater than 10 μm, i.e., on the order of approximately12 μm to 14 μm.

FIG. 7 illustrates a complete two-dimensional pixel array 95 accordingto the design of Milgrew et al., together with accompanying row andcolumn decoder circuitry and measurement readout circuitry. The array 95includes sixteen columns 85 ₁ through 85 ₁₆ of pixels, each columnhaving sixteen pixels as discussed above in connection with FIG. 3(i.e., a 16 pixel by 16 pixel array). A row decoder 92 provides sixteenpairs of complementary row select signals, wherein each pair of rowselect signals simultaneously enables one pixel in each column 85 ₁through 85 ₁₆ to provide a set of column output signals from the array95 based on the respective source voltages V_(S1) through V_(S16) of theenabled row of ISFETs. The row decoder 92 is implemented as aconventional four-to-sixteen decoder (i.e., a four-bit binary inputROW₁-ROW₄ to select one of 2⁴ outputs). The set of column output signalsV_(S1) through V_(S16) for an enabled row of the array is applied toswitching logic 96, which includes sixteen transmission gates S1 throughS16 (one transmission gate for each output signal). As above, eachtransmission gate of the switching logic 96 is implemented using ap-channel MOSFET and an n-channel MOSFET to ensure a sufficient dynamicrange for each of the output signals V_(S1) through V_(S16). The columndecoder 94, like the row decoder 92, is implemented as a conventionalfour-to-sixteen decoder and is controlled via the four-bit binary inputCOL₁-COL₄ to enable one of the transmission gates S1 through S16 of theswitching logic 96 at any given time, so as to provide a single outputsignal V_(S) from the switching logic 96. This output signal V_(S) isapplied to a 10-bit analog to digital converter (ADC) 98 to provide adigital representation D₁-D₁₀ of the output signal V_(S) correspondingto a given pixel of the array.

As noted earlier, individual ISFETs and arrays of ISFETs similar tothose discussed above have been employed as sensing devices in a varietyof applications involving chemistry and biology. In particular, ISFETshave been employed as pH sensors in various processes involving nucleicacids such as DNA. Some examples of employing ISFETs in variouslife-science related applications are given in the followingpublications, each of which is incorporated herein by reference: MassimoBarbaro, Annalisa Bonfiglio, Luigi Raffo, Andrea Alessandrini, PaoloFacci and Imrich Barak, “Fully electronic DNA hybridization detection bya standard CMOS biochip,” Sensors and Actuators B: Chemical, Volume 118,Issues 1-2, 2006, pp. 41-46; Toshinari Sakurai and Yuzuru Husimi,“Real-time monitoring of DNA polymerase reactions by a micro ISFET pHsensor,” Anal. Chem., 64(17), 1992, pp 1996-1997; S. Purushothaman, C.Toumazou, J. Georgiou, “Towards fast solid state DNA sequencing,”Circuits and Systems, vol. 4, 2002, pp. IV-169 to IV-172; S.Purushothaman, C. Toumazou, C. P. Ou, “Protons and single nucleotidepolymorphism detection: A simple use for the Ion Sensitive Field EffectTransistor,” Sensors and Actuators B: Chemical, Vol. 114, no. 2, 2006,pp. 964-968; A. L. Simonian, A. W. Flounders, J. R. Wild, “FET-BasedBiosensors for The Direct Detection of Organophosphate Neurotoxins,”Electroanalysis, Vol. 16, No. 22, 2004, pp. 1896-1906; C. Toumazou, S.Purushothaman, “Sensing Apparatus and Method,” United States PatentApplication 2004-0134798, published Jul. 15, 2004; and T. W. Koo, S.Chan, X. Su, Z. Jingwu, M. Yamakawa, V. M. Dubin, “Sensor Arrays andNucleic Acid Sequencing Applications,” United States Patent Application2006-0199193, published Sep. 7, 2006.

In general, the development of rapid and sensitive nucleic acidsequencing methods utilizing automated DNA sequencers has significantlyadvanced the understanding of biology. The term “sequencing” refers tothe determination of a primary structure (or primary sequence) of anunbranched biopolymer, which results in a symbolic linear depictionknown as a “sequence” that succinctly summarizes much of theatomic-level structure of the sequenced molecule. “DNA sequencing”particularly refers to the process of determining the nucleotide orderof a given DNA fragment. Analysis of entire genomes of viruses,bacteria, fungi, animals and plants is now possible, but such analysisgenerally is limited due to the cost and throughput of sequencing. Morespecifically, present conventional sequencing methods are limited interms of the accuracy of the sequence, the length of individualtemplates that can be sequenced, the cost of the sequence, and the rateof sequence determination.

Despite improvements in sample preparation and sequencing technologies,none of the present conventional sequencing strategies, including thoseto date that may involve ISFETs, has provided the cost reductionsrequired to increase throughput to levels required for analysis of largenumbers of individual human genomes. It is necessary to sequence a largenumber of individual genomes to understand the genetic basis of diseaseand aging. In addition, a large number of cancers will need to besequenced to understand the somatic changes underlying cancer. Somerecent efforts have made significant gains in both the ability toprepare genomes for sequencing and to sequence large numbers oftemplates simultaneously. However, these and other efforts are stilllimited by the relatively large size of the reaction volumes needed toprepare templates that are detectable by these systems, as well as theneed for special nucleotide analogues, and complex enzymatic orfluorescent methods to read out the bases.

SUMMARY

Applicants have recognized and appreciated that large arrays of ISFETsmay be particularly configured and employed to facilitate DNA sequencingtechniques based on monitoring changes in chemical processes relating toDNA synthesis. More generally, Applicants have recognized andappreciated that large arrays of chemically-sensitive FETs may beemployed to detect and measure concentrations/levels of a variety ofanalytes (e.g., hydrogen ions, other ions, non-ionic molecules orcompounds, binding events, etc.) in a host of chemical and/or biologicalprocesses (chemical reactions, cell cultures, neural activity, nucleicacid sequencing, etc.) in which valuable information may be obtainedbased on such analyte measurements.

Accordingly, various embodiments of the present disclosure is directedgenerally to inventive methods and apparatus relating to large scale FETarrays for measuring one or more analytes. In the various embodimentsdisclosed herein, FET arrays include multiple “chemFETs,” orchemically-sensitive field-effect transistors, that act as chemicalsensors. An ISFET, as discussed above, is a particular type of chemFETthat is configured for ion detection, and ISFETs may be employed invarious embodiments disclosed herein. Other types of chemFETscontemplated by the present disclosure include ENFETs, which areconfigured for sensing of specific enzymes. It should be appreciated,however, that the present disclosure is not limited to ISFETs andENFETs, but more generally relates to any FET that is configured forsome type of chemical sensitivity.

According to yet other embodiments, the present disclosure is directedgenerally to inventive methods and apparatus relating to the delivery tothe above-described large scale chemFET arrays of appropriate chemicalsamples to evoke corresponding responses. The chemical samples maycomprise (liquid) analyte samples in small reaction volumes, tofacilitate high speed, high-density determination of chemical (e.g., ionor other constituent) concentration or other measurements on theanalyte.

For example, some embodiments are directed to a “very large scale”two-dimensional chemFET sensor array (e.g., greater than 256 k sensors),in which one or more chemFET-containing elements or “pixels”constituting the sensors of such an array are configured to monitor oneor more independent chemical reactions or events occurring in proximityto the pixels of the array. In some exemplary implementations, the arraymay be coupled to one or more microfluidics structures that form one ormore reaction chambers, or “wells” or “microwells,” over individualsensors or groups of sensors of the array, and apparatus which deliversanalyte samples to the wells and removes them from the wells betweenmeasurements. Even when microwells are not employed, the sensor arraymay be coupled to one or more microfluidics structures for the deliveryof one or more anal ytes to the pixels and for removal of analyte(s)between measurements. AccOrdingly, inventive aspects of this disclosure,which are desired to be protected, include the various microfluidicstructures which may be employed to flow reagents/analytes to and fromthe wells or pixels, the methods of manufacture of the array of wells,methods and structures for coupling the wells with the pixels of thearray, and methods and apparatus for loading the wells with DNA-bearingbeads when the apparatus is used for DNA sequencing or related analysis.

A unique reference electrode and its coupling to the flow cell are alsoshown.

In various embodiments, an analyte of particular interest is hydrogenions, and large scale ISFET arrays according to the present disclosureare specifically configured to measure pH. In other embodiments, thechemical reactions being monitored may relate to DNA synthesisprocesses, or other chemical and/or biological processes, and chemFETarrays may be specifically configured to measure pH or one or more otheranalytes that provide relevant information relating to a particularchemical process of interest. In various aspects, the chemFET arrays arefabricated using conventional CMOS processing technologies, and areparticularly configured to facilitate the rapid acquisition of data fromthe entire array (scanning all of the pixels to obtain correspondingpixel output signals).

With respect to analyte detection and measurement, it should beappreciated that in various embodiments discussed in greater detailbelow, one or more analytes measured by a chemFET array according to thepresent disclosure may include any of a variety of chemical substancesthat provide relevant information regarding a chemical process orchemical processes of interest (e.g., binding of multiple nucleic acidstrands, binding of an antibody to an antigen, etc.). In some aspects,the ability to measure levels or concentrations of one or more analytes,in addition to merely detecting the presence of an analyte, providesvaluable information in connection with the chemical process orprocesses. In other aspects, mere detection of the presence of ananalyte or analytes of interest may provide valuable information.

A chemFET array according to various inventive embodiments of thepresent disclosure may be configured for sensitivity to any one or moreof a variety of analytes/chemical substances. In one embodiment, one ormore chemFETs of an array may be particularly configured for sensitivityto one or more analytes representing one or more binding events (e.g.,associated with a nucleic acid sequencing process), and in otherembodiments different chemFETs of a given array may be configured forsensitivity to different analytes. For example, in one embodiment, oneor more sensors (pixels) of the array may include a first type ofchemFET configured to be chemically sensitive to a first analyte, andone or more other sensors of the array may include a second type ofchemFET configured to be chemically sensitive to a second analytedifferent from the first analyte. In one exemplary implementation, thefirst analyte may represent a first binding event associated with anucleic acid sequencing process, and the second analyte may represent asecond binding event associated with the nucleic acid sequencingprocess. Of course, it should be appreciated that more than twodifferent types of chemFETs may be employed in any given array to detectand/or measure different types of analytes/binding events. In general,it should be appreciated in any of the embodiments of sensor arraysdiscussed herein that a given sensor array may be “homogeneous” andinclude chemFETs of substantially similar or identical types to detectand/or measure a same type of analyte (e.g., pH or other ionconcentration), or a sensor array may be “heterogeneous” and includechemFETs of different types to detect and/or measure different analytes.

In yet other aspects, Applicants have specifically improved upon theISFET array design of Milgrew et al. discussed above in connection withFIGS. 1-7, as well as other conventional ISFET array designs, so as tosignificantly reduce pixel size, and thereby increase the number ofpixels of a chemFET array for a given semiconductor die size (i.e.,increase pixel density). In various embodiments, this increase in pixeldensity to is accomplished while at the same time increasing thesignal-to-noise ratio (SNR) of output signals corresponding torespective measurements relating to monitored chemical processes, andthe speed with which such output signals may be read from the array. Inparticular, Applicants have recognized and appreciated that by relaxingrequirements for chemFET linearity and focusing on a more limitedmeasurement output signal range (e.g., output signals corresponding to apH range of from approximately 7 to 9 rather than 1 to 14), individualpixel complexity and size may be significantly reduced, therebyfacilitating the realization of very large scale dense chemFET arrays.Applicants have also recognized and appreciated that alternative lesscomplex approaches to pixel selection in an chemFET array (e.g.,alternatives to the row and column decoder approach employed in thedesign of Milgrew et al. as shown in FIG. 7, whose complexity scaleswith array size) facilitate rapid acquisition of data from significantlylarge and dense arrays.

With respect to chemFET array fabrication, Applicants have furtherrecognized and appreciated that various techniques employed in aconventional CMOS fabrication process, as well as variouspost-fabrication processing steps (wafer handling, cleaning, dicing,packaging, etc.), may in some instances adversely affect performance ofthe resulting chemFET array. For example, with reference again to FIG.1, one potential issue relates to trapped charge that may be induced inthe gate oxide 65 during etching of metals associated with the floatinggate structure 70, and how such trapped charge may affect chemFETthreshold voltage V_(TH). Another potential issue relates to thedensity/porosity of the chemFET passivation layer (e.g., see ISFETpassivation layer 72 in FIG. 1) resulting from low-temperature materialdeposition processes commonly employed in aluminum metal-based CMOSfabrication. While such low-temperature processes generally provide anadequate passivation layer for conventional CMOS devices, they mayresult in a somewhat low-density and porous passivation layer which maybe potentially problematic for chemFETs in contact with an analytesolution; in particular, a low-density porous passivation layer overtime may absorb and become saturated with analytes or other substancesin the solution, which may in turn cause an undesirable time-varyingdrift in the chemFETs threshold voltage V_(TH). This phenomenon in turnimpedes accurate measurements of one or more particular analytes ofinterest. In view of the foregoing, other inventive embodimentsdisclosed herein relate to methods and apparatus which mitigatepotentially adverse effects on chemFET performance that may arise fromvarious aspects of fabrication and post-fabrication processing/handlingof chemFET arrays.

Accordingly, one embodiment of the present invention is directed to anapparatus, comprising an array of CMOS-fabricated sensors, each sensorcomprising one chemically-sensitive field effect transistor (chemFET)and occupying an area on a surface of the array of ten micrometers byten micrometers or less.

Another embodiment is directed to a sensor array, comprising atwo-dimensional array of electronic sensors including at least 512 rowsand at least 512 columns of the electronic sensors, each sensorcomprising one chemically-sensitive field effect transistor (chemFET)configured to provide at least one output signal representing a presenceand/or concentration of an analyte proximate to a surface of thetwo-dimensional array.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising one chemically-sensitivefield effect transistor (chemFET). The array of CMOS-fabricated sensorsincludes more than 256 sensors, and a collection of chemFET outputsignals from all chemFETs of the array constitutes a frame of data. Theapparatus further comprises control circuitry coupled to the array andconfigured to generate at least one array output signal to providemultiple frames of data from the array at a frame rate of at least 1frame per second. In one aspect, the frame rate may be at least 10frames per second. In another aspect, the frame rate may be at least 20frames per second. In yet other aspects, the frame rate may be at least30, 40, 50, 70 or up to 100 frames per second.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The chemFET comprises a floating gatestructure, and a source and a drain having a first semiconductor typeand fabricated in a region having a second semiconductor type, whereinthere is no electrical conductor that electrically connects the regionhaving the second semiconductor type to either the source or the drain.

Another embodiment is directed to an apparatus, comprising an array ofelectronic sensors, each sensor consisting of three field effecttransistors (FETs) including one chemically-sensitive field effecttransistor (chemFET).

Another embodiment is directed to an apparatus, comprising an array ofelectronic sensors, each sensor comprising three or fewer field effecttransistors (FETs), wherein the three or fewer FETs includes onechemically-sensitive field effect transistor (chemFET).

Another embodiment is directed to an apparatus, comprising an array ofelectronic sensors, each sensor comprising a plurality of field effecttransistors (FETs) including one chemically-sensitive field effecttransistor (chemFET), and a plurality of electrical conductorselectrically connected to the plurality of FETs, wherein the pluralityof FETs are arranged such that the plurality of electrical conductorsincludes no more than four conductors traversing an area occupied byeach sensor and interconnecting multiple sensors of the array.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a plurality of fieldeffect transistors (FETs) including one chemically-sensitive fieldeffect transistor (chemFET), wherein all of the FETs in each sensor areof a same channel type and implemented in a single semiconductor regionof an array substrate.

Another embodiment is directed to a sensor array, comprising a pluralityof electronic sensors arranged in a plurality of rows and a plurality ofcolumns. Each sensor comprises one chemically-sensitive field effecttransistor (chemFET) configured to provide at least one output signalrepresenting a presence and/or a concentration of an analyte proximateto a surface of the array. For each column of the plurality of columns,the array further comprises column circuitry configured to provide aconstant drain current and a constant drain-to-source voltage torespective chemFETs in the column, the column circuitry including twooperational amplifiers and a diode-connected FET arranged in a Kelvinbridge configuration with the respective chemFETs to provide theconstant drain-to-source voltage.

Another embodiment is directed to a sensor array, comprising a pluralityof electronic sensors arranged in a plurality of rows and a plurality ofcolumns. Each sensor comprises one chemically-sensitive field effecttransistor (chemFET) configured to provide at least one output signalrepresenting a concentration of ions in an analyte proximate to asurface of the array. The array further comprises at least one rowselect shift register to enable respective rows of the plurality ofrows, and at least one column select shift register to acquire chemFEToutput signals from respective columns of the plurality of columns.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The chemFET comprises a floating gatestructure, and a source and a drain having a first semiconductor typeand fabricated in a region having a second semiconductor type, whereinthere is no electrical conductor that electrically connects the regionhaving the second semiconductor type to either the source or the drain.The array includes a two-dimensional array of at least 512 rows and atleast 512 columns of the CMOS-fabricated sensors. Each sensor consistsof three field effect transistors (FETs) including the chemFET, and eachsensor includes a plurality of electrical conductors electricallyconnected to the three FETs. The three FETs are arranged such that theplurality of electrical conductors includes no more than four conductorstraversing an area occupied by each sensor and interconnecting multiplesensors of the array. All of the FETs in each sensor are of a samechannel type and implemented in a single semiconductor region of anarray substrate. A collection of chemFET output signals from allchemFETs of the array constitutes a frame of data. The apparatus furthercomprises control circuitry coupled to the array and configured togenerate at least one array output signal to provide multiple frames ofdata from the array at a frame rate of at least 20 frames per second.

Another embodiment is directed to a method for processing an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The method comprises: A) dicing asemiconductor wafer including the array to form at least one dicedportion including the array; and B) performing a forming gas anneal onthe at least one diced portion.

Another embodiment is directed to a method for processing an array ofCMOS-fabricated sensors. Each sensor comprises a chemically-sensitivefield effect transistor (chemFET) having a chemically-sensitivepassivation layer of silicon nitride and/or silicon oxynitride depositedvia plasma enhanced chemical vapor deposition (PECVD). The methodcomprises: A) depositing at least one additional passivation material onthe chemically-sensitive passivation layer so as to reduce a porosityand/or increase a density of the passivation layer.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with a chemical-sensitive field effect transistor(chemFET) array comprising at least one chemFET for each reactionchamber, and wherein each of the template nucleic acids is hybridized toa sequencing primer and is bound to a polymerase, synthesizing a newnucleic acid strand by incorporating one or more known nucleotidetriphosphates sequentially at the 3′ end of the sequencing primer,detecting the incorporation of the one or more known nucleotidetriphosphates by a change in current at the at least one chemFET.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with a chemical-sensitive field effect transistor(chemFET) array comprising at least one chemFET for each reactionchamber, and wherein each of the template nucleic acids is hybridized toa sequencing primer and is bound to a polymerase, synthesizing a newnucleic acid strand by incorporating one or more known nucleotidetriphosphates sequentially at the 3′ end of the sequencing primer,detecting the incorporation of the one or more known nucleotidetriphosphates by a change in current at the at least one chemFET,wherein the chemFET array is any of the foregoing arrays.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with an chemical-sensitive field effecttransistor (chemFET) array comprising at least one chemFET for eachreaction chamber, and wherein each of the template nucleic acids ishybridized to a sequencing primer and is bound to a polymerase,synthesizing a new nucleic acid strand by incorporating one or moreknown nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, detecting the incorporation of the one or more knownnucleotide triphosphates by the generation of sequencing reactionbyproduct, wherein (a) the chemFET array comprises more than 256sensors, or (b) a center-to-center distance between adjacent reactionchambers (or “pitch”) is 1-10 μm.

Various embodiments apply equally to the methods disclosed herein andthey are recited once for brevity. In some embodiments, thecenter-to-center distance between adjacent reaction chambers is about2-9 μm, about 2 μm, about 5 μm, or about 9 μm. In some embodiments, thechemFET array comprises more than 256 sensors (and optionally more than256 corresponding reaction chambers (or wells), more than 10³ sensors(and optionally more than 10³ corresponding reaction chambers), morethan 10⁴ sensors (and optionally more than 10⁴ corresponding reactionchambers), more than 10⁵ sensors (and optionally more than 10⁵corresponding reaction chambers), or more than 10⁶ sensors (andoptionally more than 10⁶ corresponding reaction chambers). In someembodiments, the chemFET array comprises at least 512 rows and at least512 columns of sensors.

In some embodiments, the sequencing reaction byproduct is inorganicpyrophosphate (PPi). In some embodiments, PPi is measured directly. Insome embodiments, the PPi is measured in the absence of a PPi receptor.In some embodiments, the sequencing reaction byproduct is hydrogen ions.In some embodiments, the sequencing reaction byproduct is inorganicphosphate (Pi). In still other embodiments, the chemFET detects changesin any combination of the byproducts, optionally in combination withother parameters, as described herein.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with an chemical-sensitive field effecttransistor (chemFET) array comprising at least one chemFET for eachreaction chamber, and wherein each of the template nucleic acids ishybridized to a sequencing primer and is bound to a polymerase,synthesizing a new nucleic acid strand by incorporating one or moreknown nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, directly detecting release of inorganic pyrophosphate(PPi) as an indicator of incorporation of the one or more knownnucleotide triphosphates.

In some embodiments, the PPi is directly detected by binding to a PPireceptor immobilized on the chemFET. In some embodiments, the PPi isdirectly detected by the chemFET in the absence of a PPi receptor.

In another aspect, the invention provides a method for sequencingnucleic acids comprising fragmenting a template nucleic acid to generatea plurality of fragmented nucleic acids, attaching one strand from eachof the plurality of fragmented nucleic acids individually to beads togenerate a plurality of beads each having a single stranded fragmentednucleic acid attached thereto, delivering the plurality of beads havinga single stranded fragmented nucleic acid attached thereto to a chemFETarray having a separate reaction chamber for each sensor in the area,and wherein only one bead is situated in each reaction chamber, andperforming a sequencing reaction simultaneously in the plurality ofchambers.

In another aspect, the invention provides an apparatus comprising achemical-sensitive field effect transistor (chemFET) having disposed onits surface a PPi receptor.

In some embodiments, the PPi selective receptor is Compound 1, Compound2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound8, Compound 9 or Compound 10 as shown in FIG. 11B. In some embodiments,the chemFET is present in an array of chemFETs, each of which hasdisposed on its surface a PPi selective receptor. In some embodiments,the identical PPi selective receptors are disposed on each chemFET ofthe array. In some embodiments, the array comprises more than 256sensors. In some embodiments, the array comprises at least 512 rows andat least 512 columns of sensors. In some embodiments, the chemFET islocated at a bottom of a reaction chamber.

In another aspect, the invention provides an apparatus comprising achemical-sensitive field effect transistor (chemFET) array havingdisposed on its surface a biological array.

The biological array may be a nucleic acid array, a protein arrayincluding but not limited to an enzyme array, an antibody array and anantibody fragment array, a cell array, and the like. The chemical arraymay be an organic small molecule array, or an inorganic molecule array,but it is not so limited. The chemFET array may comprise at least 5, 10,10², 10³, 10⁴, 10⁵, 10⁶ or more sensors. The biological or chemicalarray may be arranged into a plurality of “cells” or spatially definedregions and each of these regions is situated over a different sensor inthe chemFET array, in some embodiments.

In yet another aspect, the invention provides a method for detecting anucleic acid comprising contacting a nucleic acid array disposed on achemFET array with a sample, and detecting binding of a nucleic acidfrom the sample to one or more regions on the nucleic acid array.

In another aspect, the invention provides a method for detecting aprotein comprising contacting a protein array disposed on a chemFETarray with a sample, and detecting binding of a protein from the sampleto one or more regions on the protein array.

In yet another aspect, the invention provides a method for detecting anucleic acid comprising contacting a protein array disposed on a chemFETarray with a sample, and detecting binding of a nucleic acid from thesample to one or more regions on the protein array.

In another aspect, the invention provides a method for detecting anantigen comprising contacting an antibody array disposed on a chemFETarray with a sample, and detecting binding of a antigen from the sampleto one or more regions on the antibody array.

In another aspect, the invention provides a method for detecting anenzyme substrate or inhibitor comprising contacting an enzyme arraydisposed on a chemFET array with a sample, and detecting binding of anentity from the sample to one or more regions on the enzyme array.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein. It should also be appreciated that terminologyexplicitly employed herein that also may appear in any disclosureincorporated by reference should be accorded a meaning most consistentwith the particular concepts disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead being placed upon generallyillustrating the various concepts discussed herein.

FIG. 1 illustrates a cross-section of a p-type (p-channel) ion-sensitivefield effect transistor (ISFET) fabricated using a conventional CMOSprocess.

FIG. 2 illustrates an electric circuit representation of the p-channelISFET shown in FIG. 1.

FIG. 3 illustrates one column of a two-dimensional ISFET array based onthe ISFET shown in FIG. 1.

FIG. 4 illustrates a transmission gate including a p-channel MOSFET andan n-channel MOSFET that is employed in each pixel of the array columnshown in FIG. 3.

FIG. 5 is a diagram similar to FIG. 1, illustrating a widercross-section of a portion of a substrate corresponding to one pixel ofthe array column shown in FIG. 3, in which the ISFET is shown alongsidetwo n-channel MOSFETs also included in the pixel.

FIG. 6 is a diagram similar to FIG. 5, illustrating a cross-section ofanother portion of the substrate corresponding to one pixel of the arraycolumn shown in FIG. 3, in which the ISFET is shown alongside thep-channel MOSFET of the transmission gate shown in FIG. 4.

FIG. 7 illustrates an example of a complete two-dimensional ISFET pixelarray based on the column design of FIG. 3, together with accompanyingrow and column decoder circuitry and measurement readout circuitry.

FIG. 8 generally illustrates a nucleic acid processing system comprisinga large scale chemFET array, according to one inventive embodiment ofthe present disclosure.

FIG. 9 illustrates one column of an chemFET array similar to that shownin FIG. 8, according to one inventive embodiment of the presentdisclosure.

FIG. 9A illustrates a circuit diagram for an exemplary amplifieremployed in the array column shown in FIG. 9, and FIG. 9B is a graph ofamplifier bias vs. bandwidth, according to one inventive embodiment ofthe present disclosure.

FIG. 10 illustrates a top view of a chip layout design for a pixel ofthe column of an chemFET array shown in FIG. 9, according to oneinventive embodiment of the present disclosure.

FIG. 11A shows a composite cross-sectional view along the line I-I ofthe pixel shown in FIG. 10, including additional elements on the righthalf of FIG. 10 between the lines II-II and III-III, illustrating alayer-by-layer view of the pixel fabrication according to one inventiveembodiment of the present disclosure.

FIG. 11B provides the chemical structures of ten PPi receptors(compounds 1 through 10).

FIG. 11C is a schematic of a synthesis protocol for compound 4 from FIG.11 B.

FIG. 11D is a schematic illustrating a variety of chemistries that canbe applied to the passivation layer in order to bind molecularrecognition compounds (such as but not limited to PPi receptors).

FIGS. 12A through 12L provide top views of each of the fabricationlayers shown in FIG. 11A, according to one inventive embodiment of thepresent disclosure.

FIG. 13 illustrates a block diagram of an exemplary CMOS IC chipimplementation of an chemFET sensor array similar to that shown in FIG.8, based on the column and pixel designs shown in FIGS. 9-12, accordingto one inventive embodiment of the present disclosure.

FIG. 14 illustrates a row select shift register of the array shown inFIG. 13, according to one inventive embodiment of the presentdisclosure.

FIG. 15 illustrates one of two column select shift registers of thearray shown in FIG. 13, according to one inventive embodiment of thepresent disclosure.

FIG. 16 illustrates one of two output drivers of the array shown in FIG.13, according to one inventive embodiment of the present disclosure.

FIG. 17 illustrates a block diagram of the chemFET sensor array of FIG.13 coupled to an array controller, according to one inventive embodimentof the present disclosure.

FIG. 18 illustrates an exemplary timing diagram for various signalsprovided by the array controller of FIG. 17, according to one inventiveembodiment of the present disclosure.

FIGS. 19-20 illustrate block diagrams of alternative CMOS IC chipimplementations of chemFET sensor arrays, according to other inventiveembodiments of the present disclosure.

FIG. 20A illustrates a top view of a chip layout design for a pixel ofthe chemFET array shown in FIG. 20, according to another inventiveembodiment of the present disclosure.

FIGS. 21-23 illustrate block diagrams of additional alternative CMOS ICchip implementations of chemFET sensor arrays, according to otherinventive embodiments of the present disclosure.

FIG. 24 illustrates the pixel design of FIG. 9 implemented with ann-channel chemFET and accompanying n-channel MOSFETs, according toanother inventive embodiment of the present disclosure.

FIGS. 25-27 illustrate alternative pixel designs and associated columncircuitry for chemFET arrays according to other inventive embodiments ofthe present disclosure.

FIGS. 28A and 28B are isometric illustrations of portions of microwellarrays as employed herein, showing round wells and rectangular wells, toassist three-dimensional visualization of the array structures.

FIG. 29 is a diagrammatic depiction of a top view of one corner (i.e.,the lower left corner) of the layout of a chip showing an array ofindividual ISFET sensors on a CMOS die.

FIG. 30 is an illustration of an example of a layout for a portion of a(typically chromium) mask for a one-sensor-per-well embodiment of theabove-described sensor array, corresponding to the portion of the dieshown in FIG. 29.

FIG. 31 is a corresponding layout for a mask for a 4-sensors-per-wellembodiment.

FIG. 32 is an illustration of a second mask used to mask an area whichsurrounds the array, to build a collar or wall (or basin, using thatterm in the geological sense) of resist which surrounds the active arrayof sensors on a substrate, as shown in FIG. 33A.

FIG. 33 is an illustration of the resulting basin.

FIG. 33A is an illustration of a three-layer PCM process for making themicrowell array.

FIGS. 34-37 diagrammatically illustrate a first example of a suitableexperiment apparatus incorporating a fluidic interface with the sensorarray, with FIG. 35 providing a cross-section through the FIG. 34apparatus along section line 35-35′ and FIG. 36 expanding part of FIG.35, in perspective, and FIG. 37 further expanding a portion of thestructure to make the fluid flow more visible:

FIG. 38 is a diagrammatic illustration of a substrate with an etchedphotoresist layer beginning the formation of an example flow cell of acertain configuration.

FIGS. 39-41 are diagrams of masks suitable for producing a firstconfiguration of flow cell consistent with FIG. 38.

FIGS. 42-54 and 57-58 are pairs of partly isometric, sectional views ofexample apparatus and enlargements, showing ways of introducing areference electrode into, and forming, a flow cell and flow chamber,using materials such as plastic and PDMS.

FIGS. 55 and 56 are schematic, cross-sectional views of two-layer glass(or plastic) arrangements for manufacturing fluidic apparatus formounting onto a chip for use as taught herein.

FIGS. 59A-59C are illustrations of the pieces for two examples oftwo-piece injection molded parts for forming a flow cell.

FIG. 60 is a schematic illustration, in cross-section, for introducing astainless steel capillary tube as an electrode, into a downstream portof a flow cell such as the flow cells of FIGS. 59A-59C, or other flowcells.

FIG. 61 is a schematic illustrating the incorporation of a dNTP into asynthesized nucleic acid strand with concomitant release of inorganicpyrophosphate (PPi).

FIGS. 62-70 illustrate bead loading into the microfluidic arrays of theinvention.

FIG. 71A is a screen capture showing pixels with signal occurring afterdATP was added (first) resulting in a 4 base extension in template 4(see Tables 1 and 2) (left panel) and a plot of voltage versus frame (ortime) for the arrowed pixels (right panel).

FIG. 71B is a screen capture showing pixels with signal occurring afterdCTP was added next resulting in a 4 base extension in template 1 (seeTables 1, 2) (left panel) and a plot of voltage versus frame (or time)for the arrowed pixels (right panel).

FIG. 71C is a screen capture showing pixels with signal occurring afterdGTP was added next resulting in extension of templates 1, 2 and 4 (seeTables 1, 2) (left panel) and a plot of voltage versus frame (or time)for the arrowed pixels (right panel).

FIG. 71D is a screen capture showing pixels with signal occurring afterdTTP was added and run-off occurred (due the presence of all 4 dNTP) inall 4 templates (see Tables 1, 2) (left panel) and a plot of voltageversus frame (or time) for the arrowed pixels (right panel).

DETAILED DESCRIPTION

Following below are more detailed descriptions of various conceptsrelated to, and embodiments of, inventive methods and apparatus relatingto large scale chemFET arrays for analyte measurements. It should beappreciated that various concepts introduced above and discussed ingreater detail below may be implemented in any of numerous ways, as thedisclosed concepts are not limited to any particular manner ofimplementation. Examples of specific implementations and applicationsare provided primarily for illustrative purposes.

Various inventive embodiments according to the present disclosure aredirected at least in part to a semiconductor-based/microfluidic hybridsystem that combines the power of microelectronics with thebiocompatibility of a microfluidic system. In some examples below, themicroelectronics portion of the hybrid system is implemented in CMOStechnology for purposes of illustration. It should be appreciated,however, that the disclosure is not intended to be limiting in thisrespect, as other semiconductor-based technologies may be utilized toimplement various aspects of the microelectronics portion of the systemsdiscussed herein.

One embodiment disclosed herein is directed to a large sensor array(e.g., a two-dimensional array) of chemically-sensitive field effecttransistors (chemFETs), wherein the individual chemFET sensor elementsor “pixels” of the array are configured to detect analyte concentrationchanges in a host of chemical and/or biological processes (chemicalreactions, cell cultures, neural activity, nucleic acid sequencingprocesses, etc.) occurring in proximity to the array. Examples ofchemFETs contemplated by various embodiments discussed in greater detailbelow include, but are not limited to, ion-sensitive field effecttransistors (ISFETs) and enzyme-sensitive field effect transistors(ENFETs). In one exemplary implementation, one or more microfluidicstructures is/are fabricated above the chemFET sensor array to providefor containment and/or confinement of a chemical reaction in which ananalyte of interest may be produced. For example, in one implementation,the microfluidic structure(s) may be configured as one or more “wells”(e.g., small reaction chambers) disposed above one or more sensors ofthe array, such that the one or more sensors over which a given well isdisposed detect and measure analyte concentration in the given well.

In some embodiments, such a chemFET array/microfluidics hybrid structuremay be used to analyze solution(s)/material(s) of interest containingnucleic acids. For example, such structures may be employed to processnucleic acids in a multitude of ways that utilize sequencing of nucleicacids. In various aspects, such sequencing can be performed to determinethe identity of a sequence of nucleic acids, for single nucleotidepolymorphism detection in nucleic acid fragments, for nucleic acidexpression profiling (comparing the nucleic acid expression profilebetween two or more states—e.g., comparing between diseased and normaltissue or comparing between untreated tissue and tissue treated withdrug, enzymes, radiation or chemical treatment), for haplotyping(comparing genes or variations in genes on each of the two allelespresent in a human subject), for karyotyping (diagnostically comparingone or more genes in a test tissue—typically from an embryo/fetus priorto conception to detect birth defects—with the same genes from “normal”karyotyped subjects), and for genotyping (comparing one or more genes ina first individual of a species with the same genes in other individualsof the same species). It should be appreciated, however, that while someillustrative examples of the concepts disclosed herein are applied inthe context of nucleic acid processing, application of the conceptsdisclosed herein relating to chemFET sensor arrays is not limited tothese examples.

FIG. 8 generally illustrates a nucleic acid processing system 1000comprising a large scale chemFET array, according to one inventiveembodiment of the present disclosure. In the discussion that follows,the chemFET sensors of the array are described for purposes ofillustration as ISFETs configured for sensitivity to hydrogen ionconcentration. However, it should be appreciated that the presentdisclosure is not limited in this respect, and that in'any of theembodiments discussed herein in which ISFETs are employed as anillustrative example, other types of chemFETs may be similarly employedin alternative embodiments, as discussed in further detail below. In oneaspect, the system 1000 includes a semiconductor/microfluidics hybridstructure 300 comprising an ISFET sensor array 100 and a microfluidicsflow cell 200. In another aspect, the flow cell 200 is configured tofacilitate the sequencing of a nucleic acid template disposed in theflow cell via the controlled admission to the flow cell of a number ofsequencing reagents 272 (e.g., bases dATP, dCTP, dGTP, dTTP and otherreagents). As illustrated in FIG. 8, the admission of the sequencingreagents to the flow cell 200 may be accomplished via one or more valves270 and one or more pumps 274 that are controlled by computer 260.

In the system 1000 of FIG. 8, according to one embodiment the ISFETsensor array 100 monitors pH changes occurring in different portions ofthe flow cell 200 due to chemical reactions between one or more of thebases constituting the sequencing reagents 272 and the nucleic acidtemplate. In other embodiments discussed in greater detail below, theFET sensor array may be particularly configured for sensitivity to otheranalytes that may provide relevant information about the chemicalreactions of interest. Via an array controller 250 (also under operationof the computer 260), the ISFET array may be controlled so as to acquiredata relating to analyte measurements, and collected data may beprocessed by the computer 260 to yield meaningful information associatedwith the processing of the nucleic acid template. For example, in oneimplementation, pH change generally is proportional to the number of aparticular type of base (e.g., one of dATP, dCTP, dGTP, dTTP) added tothe nucleic acid template. Such a pH change may be represented by achange in output voltage of one or more ISFETs of the array 100 inproximity to the reaction(s) between a given type of base and thetemplate. Thus, the magnitude of the voltage change in an output signalof a given pixel of the array may be used to determine the number ofbases of a particular type added to the template disposed in the flowcell above the given pixel.

In one aspect, the flow cell 200 of the system 1000 shown in FIG. 8 maycomprise a number of wells (not shown in FIG. 8) disposed abovecorresponding sensors of the ISFET array 100. A number of techniques maybe used to admit the various processing materials to the wells of such aflow cell. For example, the flow cell first may be loaded with a nucleicacid template to be sequenced by centrifuging into the wells “beads”containing the nucleic acid template; alternatively, such beads mayenter the wells by gravity. In another example, instead of employingbeads, the wells can be coated with a set of primer pairs, and thenucleic acid template provided to the flow cell with adapters thatcomplement the primer pairs (immobilization materials can be added tothe sensor array 100, or to individual dies as part of the chippackaging, or immediately before the processing of the nucleic acid).Other methods involving solgels may be used to immobilize a nucleic acidtemplate near the surface of the ISFET array 100.

Once a nucleic acid template is loaded into respective wells of the flowcell 200, bridge amplification can then be performed in the wells, theproduct denatured, and sequencing by synthesis or ligation thenperformed. Other methods of amplification in the wells (with theproducts captured in the wells) are envisioned, including rolling circleamplification, or other strategies using isothermal or non-isothermalamplification techniques such as PCR. As illustrated in FIG. 8, reagentsincluding bases may be admitted to the flow cell (e.g., via the computercontrolled valve 270 and pumps 274) and diffuse into the wells, orreagents may be added to the flow cell by other means such as an inkjet. In yet another example, the flow cell 200 may not contain anywells, and diffusion properties of the reagents may be exploited tolimit cross-talk between respective sensors of the ISFET array 100.

In sum, the flow cell 200 in the system of FIG. 8 may be configured in avariety of manners to provide one or more analytes in proximity to theISFET array 100; for example, a nucleic acid template (DNA) may bedirectly attached or applied in suitable proximity to one or more pixelsof the sensor array 100, or on a support material (e.g., one or more“beads”) located above the sensor array. Processing reagents (e.g.,enzymes) can also be placed on the sensors directly, or on one or moresolid supports in proximity to the array, and the device used withoutwells or beads for a number of biosensor applications where the enzymesresult in a sensor-detectable product (e.g., ion concentration change).

With respect to the ISFET array 100 of the system 1000 shown in FIG. 8,in one embodiment the array 100 is implemented as an integrated circuitdesigned and fabricated using standard CMOS processes (e.g., 0.35micrometer process, 0.18 micrometer process), comprising all the sensorsand electronics needed to monitor/measure one or more analytes. Withreference again to FIG. 1, one or more reference electrodes 76 to beemployed in connection with the ISFET array 100 may be placed in theflow cell 200 (e.g., disposed in “unused” wells of the flow cell) orotherwise exposed to a reference (e.g., one or more of the sequencingreagents 172) to establish a base line against which changes in analyteconcentration proximate to respective ISFETs of the array 100 arecompared. The reference electrode(s) 76 may be electrically coupled tothe array 100, the array controller 250 or directly to the computer 260to facilitate analyte measurements based on voltage signals obtainedfrom the array 100; in some implementations, the reference electrode(s)may be coupled to an electric ground or other predetermined potential,or the reference electrode voltage may be measured with respect toground, to establish an electric reference for ISFET output signalmeasurements, as discussed further below.

The ISFET array 100 is not limited to any particular size, as one- ortwo-dimensional arrays, including as few as two to 256 pixels (e.g., 16by 16 pixels in a two-dimensional implementation) or as many as 54mega-pixels (e.g., 7400 by 7400 pixels in a two-dimensionalimplementation) or even greater may be fabricated and employed forvarious chemical/biological analysis purposes pursuant to the conceptsdisclosed herein. In one embodiment of the exemplary system shown inFIG. 8, the individual ISFET sensors of the array may be configured forsensitivity to hydrogen ions; however, it should also be appreciatedthat the present disclosure is not limited in this respect, asindividual sensors of an ISFET sensor array may be particularlyconfigured for sensitivity to other types of ion concentrations for avariety of applications (materials sensitive to other ions such assodium, silver, iron, bromine, iodine, calcium, and nitrate, forexample, are known).

More generally, a chemFET array according to various embodiments of thepresent disclosure may be configured for sensitivity to any one or moreof a variety of analytes/chemical substances. In one embodiment, one ormore chemFETs of an array may be particularly configured for sensitivityto one or more analytes representing one or more binding events (e.g.,associated with a nucleic acid sequencing process), and in otherembodiments different chemFETs of a given array may be configured forsensitivity to different analytes. For example, in one embodiment, oneor more sensors (pixels) of the array may include a first type ofchemFET configured to be chemically sensitive to a first analyte, andone or more other sensors of the array may include a second type ofchemFET configured to be chemically sensitive to a second analytedifferent from the first analyte. In one exemplary implementation, thefirst analyte may represent a first binding event associated with anucleic acid sequencing process, and the second analyte may represent asecond binding event associated with the nucleic acid sequencingprocess. Of course, it should be appreciated that more than twodifferent types of chemFETs may be employed in any given array to detectand/or measure different types of analytes/binding events. In general,it should be appreciated in any of the embodiments of sensor arraysdiscussed herein that a given sensor array may be “homogeneous” andinclude chemFETs of substantially similar or identical types to detectand/or measure a same type of analyte (e.g., pH or other ionconcentration), or a sensor array may be “heterogeneous” and includechemFETs of different types to detect and/or measure different analytes.For simplicity of discussion, again the example of an ISFET is discussedbelow in various embodiments of sensor arrays, but the presentdisclosure is not limited in this respect, and several other options foranalyte sensitivity are discussed in further detail below (e.g., inconnection with FIG. 11A).

In exemplary implementations based on 0.35 micrometer CMOS processingtechniques (or CMOS processing techniques capable of smaller featuresizes), each pixel of the ISFET array 100 may include an ISFET andaccompanying enable/select components, and may occupy an area on asurface of the array of approximately ten micrometers by ten micrometers(i.e., 100 micrometers²) or less; stated differently, arrays having apitch (pixel-to-pixel spacing) on the order of 10 micrometers or lessmay be realized. An array pitch on the order of 10 micrometers or lessusing a 0.35 micrometer CMOS processing technique constitutes asignificant improvement in terms of size reduction with respect to priorattempts to fabricate ISFET arrays, which resulted in pixel sizes on theorder of at least 12 micrometers or greater.

More specifically, in some embodiments discussed further below based onthe inventive concepts disclosed herein, an array pitch of approximatelynine (9) micrometers allows an ISFET array including over 256,000 pixels(i.e., a 512 by 512 array), together with associated row and columnselect and bias/readout electronics, to be fabricated on a 7 millimeterby 7 millimeter semiconductor die, and a similar sensor array includingover four million pixels (i.e., a 2048 by 2048 array, over 4Mega-pixels) to be fabricated on a 21 millimeter by 21 millimeter die.In other examples, an array pitch of approximately 5 micrometers allowsan ISFET array including approximately 1.55 Mega-pixels (i.e., a 1348 by1152 array) and associated electronics to be fabricated on a 9millimeter by 9 millimeter die, and an ISFET sensor array including over14 Mega-pixels and associated electronics on a 22 millimeter by 20millimeter die. In yet other implementations, using a CMOS fabricationprocess in which feature sizes of less than 0.35 micrometers arepossible (e.g., 0.18 micrometer CMOS processing techniques), ISFETsensor arrays with a pitch significantly below 5 micrometers may befabricated (e.g., array pitch of 2.6 micrometers or pixel area of lessthan 8 or 9 micrometers²), providing for significantly dense ISFETarrays. Of course, it should be appreciated that pixel sizes greaterthan 10 micrometers (e.g., on the order of approximately 20, 50, 100micrometers or greater) may be implemented in various embodiments ofchemFET arrays according to the present disclosure.

In other aspects of the system shown in FIG. 8, one or more arraycontrollers 250 may be employed to operate the ISFET array 100 (e.g.,selecting/enabling respective pixels of the array to obtain outputsignals representing analyte measurements). In various implementations,one or more components constituting one or more array controllers may beimplemented together with pixel elements of the arrays themselves, onthe same integrated circuit (IC) chip as the array but in a differentportion of the IC chip, or off-chip. In connection with array control,analog-to-digital conversion of ISFET output signals may be performed bycircuitry implemented on the same integrated circuit chip as the ISFETarray, but located outside of the sensor array region (locating theanalog to digital conversion circuitry outside of the sensor arrayregion allows for smaller pitch and hence a larger number of sensors, aswell as reduced noise). In various exemplary implementations discussedfurther below, analog-to-digital conversion can be 4-bit, 8-bit, 12-bit,16-bit or other bit resolutions depending on the signal dynamic rangerequired.

Having provided a general overview of the role of a chemFET (e.g.,ISFET) array 100 in an exemplary system 1000 for measuring one or moreanalytes in connection with nucleic acid processing, following below aremore detailed descriptions of exemplary chemFET arrays according tovarious inventive embodiments of the present disclosure that may beemployed in a variety of applications including, but not limited to,nucleic acid processing. Again, for purposes of illustration, chemFETarrays according to the present disclosure are discussed below using theparticular example of an ISFET array, but other types of chemFETs may beemployed in alternative embodiments.

An noted above, various inventive embodiments disclosed hereinspecifically improve upon the ISFET array design of Milgrew et al.discussed above in connection with FIGS. 1-7, as well as other priorISFET array designs, so as to significantly reduce pixel size and arraypitch, and thereby increase the number of pixels of an ISFET array for agiven semiconductor die size (i.e., increase pixel density). In someimplementations, an increase in pixel density is accomplished while atthe same time increasing the signal-to-noise ratio (SNR) of outputsignals corresponding to respective measurements relating to one or morechemical properties of one or more analytes and the speed with whichsuch output signals may be read from the array. In particular,Applicants have recognized and appreciated that by relaxing requirementsfor ISFET linearity and focusing on a more limited signaloutput/measurement range (e.g., signal outputs corresponding to a pHrange of from approximately 7 to 9 rather than 1 to 14), individualpixel complexity and size may be significantly reduced, therebyfacilitating the realization of very large scale dense ISFET arrays.

To this end, FIG. 9 illustrates one column 102 _(j) of an ISFET array100, according to one inventive embodiment of the present disclosure, inwhich ISFET pixel design is appreciably simplified to facilitate smallpixel size. The column 102 _(j) includes n pixels, the first and last ofwhich are shown in FIG. 9 as the pixels 105 ₁ and 105 _(n). As discussedfurther below in connection with FIG. 13, a complete two-dimensionalISFET array 100 based on the column design shown in FIG. 9 includes msuch columns 102 _(j) (j=1, 2, 3, . . . m) with successive columns ofpixels generally arranged side by side.

In one aspect of the embodiment shown in FIG. 9, each pixel 105 ₁through 105 _(n) of the column 102 _(j) includes only three components,namely, an ISFET 150 (also labeled as Q1) and two MOSFET switches Q2 andQ3. The MOSFET switches Q2 and Q3 are both responsive to one of n rowselect signals ( RowSel₁ through RowSel_(n) , logic low active) so as toenable or select a given pixel of the column 102 _(j). Using pixel 105 ₁as an example that applies to all pixels of the column, the transistorswitch Q3 couples a controllable current source 106 _(j) via the line112 ₁ to the source of the ISFET 150 upon receipt of the correspondingrow select signal via the line 118 ₁. The transistor switch Q2 couplesthe source of the ISFET 150 to column bias/readout circuitry 110 _(j)via the line 114 ₁ upon receipt of the corresponding row select signal.The drain of the ISFET 150 is directly coupled via the line 116 ₁ to thebias/readout circuitry 110 _(j). Thus, only four signal lines per pixel,namely the lines 112 ₁, 114 ₁, 116 ₁ and 118 ₁, are required to operatethe three components of the pixel 105 ₁. In an array of m columns, agiven row select signal is applied simultaneously to one pixel of eachcolumn (e.g., at same positions in respective columns).

As illustrated in FIG. 9, the design for the column 102 _(j) accordingto one embodiment is based on general principles similar to thosediscussed above in connection with the column design of Milgrew et al.shown FIG. 3. In particular, the ISFET of each pixel, when enabled, isconfigured with a constant drain current I_(Dj) and a constantdrain-to-source voltage V_(DSj) to obtain an output signal V_(Sj) froman enabled pixel according to Eq. (3) above. To this end, the column 102_(j) includes a controllable current source 106 _(j), coupled to ananalog circuitry positive supply voltage VDDA and responsive to a biasvoltage VB1, that is shared by all pixels of the column to provide aconstant drain current I_(Dj) to the ISFET of an enabled pixel. In oneaspect, the current source 106 _(j) is implemented as a current mirrorincluding two long-channel length and high output impedance MOSFETs. Thecolumn also includes bias/readout circuitry 110 _(j) that is also sharedby all pixels of the column to provide a constant drain-to-sourcevoltage to the ISFET of an enabled pixel. The bias/readout circuitry 110_(j) is based on a Kelvin Bridge configuration and includes twooperational amplifiers 107A (A1) and 107B (A2) configured as bufferamplifiers and coupled to analog circuitry positive supply voltage VDDAand the analog supply voltage ground VSSA. The bias/readout circuitryalso includes a controllable current sink 108 _(j) (similar to thecurrent source 106 _(j)) coupled to the analog ground VSSA andresponsive to a bias voltage VB2, and a diode-connected MOSFET Q6. Thebias voltages VB1 and VB2 are set/controlled in tandem to provide acomplimentary source and sink current. The voltage developed across thediode-connected MOSFET Q6 as a result of the current drawn by thecurrent sink 108 _(j) is forced by the operational amplifiers to appearacross the drain and source of the ISFET of an enabled pixel as aconstant drain-source voltage V_(DSj).

By employing the diode-connected MOSFET Q6 in the bias/readout circuitry110 _(j) of FIG. 9, rather than the resistor R_(SDj) as shown in thedesign of Milgrew et al. illustrated in FIG. 3, a significant advantageis provided in a CMOS fabrication process; specifically, matchingresistors can be fabricated with error tolerances generally on the orderof ±20%, whereas MOSFET matching in a CMOS fabrication process is on theorder of ±1% or better. The degree to which the component responsiblefor providing a constant ISFET drain-to-source voltage V_(DSj) can bematched from column to column significantly affects measurement accuracy(e.g., offset) from column to column. Thus, employing the MOSFET Q6rather than a resistor appreciably mitigates measurement offsets fromcolumn-to-column. Furthermore, whereas the thermal drift characteristicsof a resistor and an ISFET may be appreciably different, the thermaldrift characteristics of a MOSFET and ISFET are substantially similar,if not virtually identical; hence, any thermal drift in MOSFET Q6virtually cancels any thermal drift from ISFET Q1, resulting in greatermeasurement stability with changes in array temperature.

In FIG. 9, the column bias/readout circuitry 110 _(j) also includessample/hold and buffer circuitry to provide an output signal V_(COLj)from the column. In particular, after one of the pixels 105 ₁ through105 _(n) is enabled or selected via the transistors Q2 and Q3 in eachpixel, the output of the amplifier 107A (A1), i.e., a buffered V_(Sj),is stored on a column sample and hold capacitor C_(sh) via operation ofa switch (e.g., a transmission gate) responsive to a column sample andhold signal COL SH. Examples of suitable capacitances for the sample andhold capacitor include, but are not limited to, a range of fromapproximately 500 fF to 2 pF. The sampled voltage is buffered via acolumn output buffer amplifier 111 j (BUF) and provided as the columnoutput signal V_(COLj). As also shown in FIG. 9, a reference voltageVREF may be applied to the buffer amplifier 111 j, via a switchresponsive to a control signal CAL, to facilitate characterization ofcolumn-to-column non-uniformities due to the buffer amplifier 111 j andthus allow post-read data correction.

FIG. 9A illustrates an exemplary circuit diagram for one of theamplifiers 107A of the bias/readout circuitry 110 _(j) (the amplifier107B is implemented identically), and FIG. 9B is a graph of amplifierbias vs. bandwidth for the amplifiers 107A and 107B. As shown in FIG.9A, the amplifier 107A employs an arrangement of multiple currentmirrors based on nine MOSFETs (M1 through M9) and is configured as aunity gain buffer, in which the amplifier's inputs and outputs arelabeled for generality as IN+ and VOUT, respectively. The bias voltageVB4 (representing a corresponding bias current) controls thetransimpedance of the amplifier and serves as a bandwidth control (i.e.,increased bandwidth with increased current). With reference again toFIG. 9, due to the sample and hold capacitor Csh, the output of theamplifier 107A essentially drives a filter when the sample and holdswitch is closed. Accordingly, to achieve appreciably high data rates,the bias voltage VB4 may be adjusted to provide higher bias currents andincreased amplifier bandwidth. From FIG. 9B, it may be observed that insome exemplary implementations, amplifier bandwidths of at least 40 MHzand significantly greater may be realized. In some implementations,amplifier bandwidths as high as 100 MHz may be appropriate to facilitatehigh data acquisition rates and relatively lower pixel sample or “dwell”times (e.g., on the order of 10 to 20 microseconds).

In another aspect of the embodiment shown in FIG. 9, unlike the pixeldesign of Milgrew et al. shown in FIG. 3, the pixels 105 ₁ through 105_(n) do not include any transmission gates or other devices that requireboth n-channel and p-channel FET components; in particular, the pixels105 ₁ through 105 _(n) of this embodiment include only FET devices of asame type (i.e., only n-channel or only p-channel). For purposes ofillustration, the pixels 105 ₁ and 105 _(n) illustrated in FIG. 9 areshown as comprising only p-channel components, i.e., two p-channelMOSFETs Q2 and Q3 and a p-channel ISFET 150. By not employing atransmission gate to couple the source of the ISFET to the bias/readoutcircuitry 110 _(j), some dynamic range for the ISFET output signal(i.e., the ISFET source voltage V_(S)) may be sacrificed. However,Applicants have recognized and appreciated that by potentially foregoingsome output signal dynamic range (and thereby potentially limitingmeasurement range for a given chemical property, such as pH), therequirement of different type FET devices (both n-channel and p-channel)in each pixel may be eliminated and the pixel component count reduced.As discussed further below in connection with FIGS. 10-12, thissignificantly facilitates pixel size reduction. Thus, in one aspect,there is a beneficial tradeoff between reduced dynamic range and smallerpixel size.

In yet another aspect of the embodiment shown in FIG. 9, unlike thepixel design of Milgrew et al., the ISFET 150 of each pixel 105 ₁through 105 _(n) does not have its body connection tied to its source(i.e., there is no electrical conductor coupling the body connection andsource of the ISFET such that they are forced to be at the same electricpotential during operation). Rather, the body connections of all ISFETsof the array are tied to each other and to a body bias voltage V_(BODY).While not shown explicitly in FIG. 9, the body connections for theMOSFETs Q2 and Q3 likewise are not tied to their respective sources, butrather to the body bias voltage V_(BODY). In one exemplaryimplementation based on pixels having all p-channel components, the bodybias voltage V_(BODY) is coupled to the highest voltage potentialavailable to the array (e.g., VDDA), as discussed further below inconnection with FIG. 17.

By not tying the body connection of each ISFET to its source, thepossibility of some non-zero source-to-body voltage V_(SB) may give riseto the “body effect,” as discussed above in connection with FIG. 1,which affects the threshold voltage V_(TH) of the ISFET according to anonlinear relationship (and thus, according to Eq. (3), may affectmeasurements of chemical properties, such as pH). However, Applicantshave recognized and appreciated that by focusing on a reduced ISFEToutput signal dynamic range, any body effect that may arise in the ISFETfrom a non-zero source-to-body to voltage may be relatively minimal.Thus, any measurement nonlinearity that may result over the reduceddynamic range may be ignored as insignificant or taken intoconsideration and compensated (e.g., via array calibration and dataprocessing techniques, as discussed further below in connection withFIG. 17). Applicants have also recognized and appreciated that by nottying each ISFET source to its body connection, all of the FETsconstituting the pixel may share a common body connection, therebyfurther facilitating pixel size reduction, as discussed further below inconnection with FIGS. 10-12. Accordingly, in another aspect, there is abeneficial tradeoff between reduced linearity and smaller pixel size.

FIG. 10 illustrates a top view of a chip layout design for the pixel 105₁ shown in FIG. 9, according to one inventive embodiment of the presentdisclosure. FIG. 11A shows a composite cross-sectional view along theline I-I of the pixel shown in FIG. 10, including additional elements onthe right half of FIG. 10 between the lines II-II and III-III,illustrating a layer-by-layer view of the pixel fabrication, and FIGS.12A through 12L provide top views of each of the fabrication layersshown in FIG. 11A (the respective images of FIGS. 12A through 12L aresuperimposed one on top of another to create the pixel chip layoutdesign shown in FIG. 10). In one exemplary implementation, the pixeldesign illustrated in FIGS. 10-12 may be realized using a standard4-metal, 2-poly, 0.35 micrometer CMOS process to provide a geometricallysquare pixel having a dimension “e” as shown in FIG. 10 of approximately9 micrometers, and a dimension “f” corresponding to the ISFET sensitivearea of approximately 7 micrometers.

In the top view of FIG. 10, the ISFET 150 (labeled as Q1 in FIG. 10)generally occupies the right center portion of the pixel illustration,and the respective locations of the gate, source and drain of the ISFETare indicated as Q1 _(G), Q1 _(S) and Q1 _(D). The MOSFETs Q2 and Q3generally occupy the left center portion of the pixel illustration; thegate and source of the MOSFET Q2 are indicated as Q2 _(G) and Q2 _(S),and the gate and source of the MOSFET Q3 are indicated as Q3 _(G) and Q3_(S). In one aspect of the layout shown in FIG. 10, the MOSFETs Q2 andQ3 share a drain, indicated as Q2/3 _(D). In another aspect, it may beobserved generally from the top view of FIG. 10 that the ISFET is formedsuch that its channel lies along a first axis of the pixel (e.g.,parallel to the line I-I), while the MOSFETs Q2 and Q3 are formed suchthat their channels lie along a second axis perpendicular to the firstaxis. FIG. 10 also shows the four lines required to operate the pixel,namely, the line 112 ₁ coupled to the source of Q3, the line 114 ₁coupled to the source of Q2, the line 116 ₁ coupled to the drain of theISFET, and the row select line 118 ₁ coupled to the gates of Q2 and Q3.With reference to FIG. 9, it may be appreciated that all pixels in agiven column share the lines 112, 114 and 116 (e.g., running verticallyacross the pixel in FIG. 10), and that all pixels in a given row sharethe line 118 (e.g., running horizontally across the pixel in FIG. 10);thus, based on the pixel design of FIG. 9 and the layout shown in FIG.10, only four metal lines need to traverse each pixel.

With reference now to the cross-sectional view of FIG. 11A, highly dopedp-type regions 156 and 158 (lying along the line I-I in FIG. 10) inn-well 154 constitute the source (S) and drain (D) of the ISFET, betweenwhich lies a region 160 of the n-well in which the ISFETs p-channel isformed below the ISFETs polysilicon gate 164 and a gate oxide 165.According to one aspect of the inventive embodiment shown in FIGS. 10and 11, all of the FET components of the pixel 105 ₁ are fabricated asp-channel FETs in the single n-type well 154 formed in a p-typesemiconductor substrate 152. This is possible because, unlike the designof Milgrew et al., 1) there is no requirement for a transmission gate inthe pixel; and 2) the ISFETs source is not tied to the n-well's bodyconnection. More specifically, highly doped n-type regions 162 provide abody connection (B) to the n-well 154 and, as shown in FIG. 10, the bodyconnection B is coupled to a metal conductor 322 around the perimeter ofthe pixel 105 ₁. However, the body connection is not directlyelectrically coupled to the source region 156 of the ISFET (i.e., thereis no electrical conductor coupling the body connection and source suchthat they are forced to be at the same electric potential duringoperation), nor is the body connection directly electrically coupled tothe gate, source or drain of any component in the pixel. Thus, the otherp-channel FET components of the pixel, namely Q2 and Q3, may befabricated in the same n-well 154.

In the composite cross-sectional view of FIG. 11A, a highly doped p-typeregion 159 is also visible (lying along the line I-I in FIG. 10),corresponding to the shared drain (D) of the MOSFETs Q2 and Q3. Forpurposes of illustration, a polysilicon gate 166 of the MOSFET Q3 alsois visible in FIG. 11A, although this gate does not lie along the linein FIG. 10, but rather “behind the plane” of the cross-section along theline I-I. However, for simplicity, the respective sources of the MOSFETsQ2 and Q3 shown in FIG. 10, as well as the gate of Q2, are not visiblein FIG. 11A, as they lie along the same axis (i.e., perpendicular to theplane of the figure) as the shared drain (if shown in FIG. 11A, theseelements would unduly complicate the composite cross-sectional view ofFIG. 11A).

Above the substrate, gate oxide, and polysilicon layers shown in FIG.11A, a number of additional layers are provided to establish electricalconnections to the various pixel components, including alternating metallayers and oxide layers through which conductive vias are formed.Pursuant to the 4-Metal CMOS process, these layers are labeled in FIG.11A as “Contact,” “Metal1,” “Via1,” “Metal2,” “Via1,” “Metal3,” “Via3,”and “Metal4.” To facilitate an understanding particularly of the ISFETelectrical connections, the composite cross-sectional view of FIG. 11Ashows additional elements of the pixel fabrication on the right side ofthe top view of FIG. 10 between the lines and III-III. With respect tothe ISFET electrical connections, the topmost metal layer 304corresponds to the ISFETs sensitive area 178, above which is disposed ananalyte-sensitive passivation layer 172. The topmost metal layer 304,together with the ISFET polysilicon gate 164 and the interveningconductors 306, 308, 312, 316, 320, 326 and 338, form the ISFETs“floating gate” structure 170, in a manner similar to that discussedabove in connection with a conventional ISFET design shown in FIG. 1. Anelectrical connection to the ISFETs drain is provided by the conductors340, 328, 318, 314 and 310 coupled to the line 116 ₁. The ISFETs sourceis coupled to the shared drain of the MOSFETs Q2 and Q3 via theconductors 334 and 336 and the conductor 324 (which lies along the lineI-I in FIG. 10). The body connections 162 to the n-well 154 areelectrically coupled to a metal conductor 322 around the perimeter ofthe pixel on the “Metal1” layer via the conductors 330 and 332.

As indicated above, FIGS. 12A through 12L provide top views of each ofthe fabrication layers shown in FIG. 11A (the respective images of FIGS.12A through 12L are superimposed one on top of another to create thepixel chip layout design shown in FIG. 10). In FIG. 12, thecorrespondence between the lettered top views of respective layers andthe cross-sectional view of FIG. 11A is as follows: A) n-type well 154;B) Implant; C) Diffusion; D) polysilicon gates 164 (ISFET) and 166(MOSFETs Q2 and Q3); E) contacts; F) Metal1; G) Via1; H) Metal2; I)Via1; J) Metal3; K) Via3; L) Metal4 (top electrode contacting ISFETgate). The various reference numerals indicated in FIGS. 12A through 12Lcorrespond to the identical features that are present in the compositecross-sectional view of FIG. 11A.

Thus, the pixel chip layout design shown in FIGS. 10, 11, and 12Athrough 12L illustrates that, according to one embodiment, FET devicesof a same type may be employed for all components of the pixel, and thatall components may be implemented in a single well. This dramaticallyreduces the area required for the pixel, thereby facilitating increasedpixel density in a given area.

In one exemplary implementation, the gate oxide 165 for the ISFET may befabricated to have a thickness on the order of approximately 75Angstroms, giving rise to a gate oxide capacitance per unit area C_(ox)of 4.5 fF/μm². Additionally, the polysilicon gate 164 may be fabricatedwith dimensions corresponding to a channel width W of 1.2 μm and achannel length L of from 0.35 to 0.6 μm (i.e., W/L ranging fromapproximately 2 to 3.5), and the doping of the region 160 may beselected such that the carrier mobility for the p-channel is 190 cm²/V·s(i.e., 1.9 E10 μm²/V·s). From Eq. (2) above, this results in an ISFETtransconductance parameter β on the order of approximately 170 to 300μA/V². In other aspects of this exemplary implementation, the analogsupply voltage VDDA is 3.3 Volts, and VB1 and VB2 are biased so as toprovide a constant ISFET drain current I_(Dj) on the order of 5 μA (insome implementations, VB1 and VB2 may be adjusted to provide draincurrents from approximately 1 μA to 20 μA). Additionally, the MOSFET Q6(see bias/readout circuitry 110 _(j) in FIG. 9) is sized to have achannel width to length ratio (e.g., W/L of approximately 50) such thatthe voltage across Q6, given I_(Dj) of 5 μA, is 800 mV (i.e.,V_(DSj)=800 mV). From Eq. (3), based on these exemplary parameters, thisprovides for pixel output voltages V_(Sj) over a range of approximately0.5 to 2.5 Volts for ISFET threshold voltage changes over a range ofapproximately 0 to 2 Volts.

With respect to the analyte-sensitive passivation layer 172 shown inFIG. 11A, in exemplary CMOS implementations the passivation layer may besignificantly sensitive to hydrogen ion concentration and may includesilicon nitride (Si₃N₄) and/or silicon oxynitride (Si₂N₂O). Inconventional CMOS processes, a passivation layer may be formed by one ormore successive depositions of these materials, and is employedgenerally to treat or coat devices so as to protect againstcontamination and increase electrical stability. The material propertiesof silicon nitride and silicon oxynitride are such that a passivationlayer comprising these materials provides scratch protection and servesas a significant barrier to the diffusion of water and sodium, which cancause device metallization to corrode and/or device operation to becomeunstable. A passivation layer including silicon nitride and/or siliconoxynitride also provides ion-sensitivity in ISFET devices, in that thepassivation layer contains surface groups that may donate or acceptprotons from an analyte solution with which they are in contact, therebyaltering the surface potential and the device threshold voltage V_(TH),as discussed above in connection with FIG. 1.

For CMOS processes involving aluminum as the metal (which has a meltingpoint of approximately 650 degrees Celsius), a silicon nitride and/orsilicon oxynitride passivation layer generally is formed viaplasma-enhanced chemical vapor deposition (PECVD), in which a glowdischarge at 250-350 degrees Celsius ionizes the constituent gases thatform silicon nitride or silicon oxynitride, creating active species thatreact at the wafer surface to form a laminate of the respectivematerials. In one exemplary process, a passivation layer having athickness on the order of approximately 1.0 to 1.5 μm may be formed byan initial deposition of a thin layer of silicon oxynitride (on theorder of 0.2 to 0.4 μm) followed by a slighting thicker deposition ofsilicon oxynitride (on the order of 0.5 μm) and a final deposition ofsilicon nitride (on the order of 0.5 μm). Because of the low depositiontemperature involved in the PECVD process, the aluminum metallization isnot adversely affected.

However, Applicants have recognized and appreciated that while a lowtemperature PECVD process provides adequate passivation for conventionalCMOS devices, the low-temperature process results in a generallylow-density and somewhat porous passivation layer, which in some casesmay adversely affect ISFET threshold voltage stability. In particular,during ISFET device operation, a low-density porous passivation layerover time may absorb and become saturated with ions from the solution,which may in turn cause an undesirable time-varying drift in the ISFETsthreshold voltage V_(TH), making accurate measurements challenging.

In view of the foregoing, in one embodiment a CMOS process that usestungsten metal instead of aluminum may be employed to fabricate ISFETarrays according to the present disclosure. The high melting temperatureof Tungsten (above 3400 degrees Celsius) permits the use of a highertemperature low pressure chemical vapor deposition (LPCVD) process(e.g., approximately 700 to 800 degrees Celsius) for a silicon nitrideor silicon oxynitride passivation layer. The LPCVD process typicallyresults in significantly more dense and less porous films for thepassivation layer, thereby mitigating the potentially adverse effects ofion absorption from the analyte solution leading to ISFET thresholdvoltage drift.

In yet another embodiment in which an aluminum-based CMOS process isemployed to fabricate ISFET arrays according to the present disclosure,the passivation layer 172 shown in FIG. 11A may comprise additionaldepositions and/or materials beyond those typically employed in aconventional CMOS process. For example, the passivation layer 172 mayinclude initial low-temperature plasma-assisted depositions (PECVD) ofsilicon nitride and/or silicon oxynitride as discussed above; forpurposes of the present discussion, these conventional depositions areillustrated in FIG. 11A as a first portion 172A of the passivation layer172. In one embodiment, following the first portion 172A, one or moreadditional passivation materials are disposed to form at least a secondportion 172B to increase density and reduce porosity of (and absorptionby) the overall passivation layer 172. While one additional portion 172Bis shown primarily for purposes of illustration in FIG. 11A, it shouldbe appreciated that the disclosure is not limited in this respect, asthe overall passivation layer 172 may comprise two or more constituentportions, in which each portion may comprise one or morelayers/depositions of same or different materials, and respectiveportions may be configured similarly or differently.

Examples of materials suitable for the second portion 172B (or otheradditional portions) of the passivation layer 172 for particularsensitivity to hydrogen ions include, but are not limited to, siliconnitride, silicon oxynitride, aluminum oxide (Al₂O₃), tantalum oxide(Ta₃O₅), tin oxide (SnO₂) and silicon dioxide (SiO₂). In one aspect, thesecond portion 172B (or other additional portions) may be deposited viaa variety of relatively low temperature processes including, but notlimited to, RF sputtering, DC magnetron sputtering, thermal or e-beamevaporation, and ion-assisted depositions. In another aspect, apre-sputtering etch process may be employed, prior to deposition of thesecond portion 172B, to remove any native oxide residing on the firstportion 172A (alternatively, a reducing environment, such as an elevatedtemperature hydrogen environment, may be employed to remove native oxideresiding on the first portion 172A). In yet another aspect, a thicknessof the second portion 172B may be on the order of approximately 0.04 μmto 0.06 μm (400 to 600 Angstroms) and a thickness of the first portionmay be on the order of 1.0 to 1.5 μm, as discussed above. In someexemplary implementations, the first portion 172A may include multiplelayers of silicon oxynitride and silicon nitride having a combinedthickness of 1.0 to 1.5 μm, and the second portion 172B may include asingle layer of either aluminum oxide or tantalum oxide having athickness of approximately 400 to 600 Angstroms. Again, it should beappreciated that the foregoing exemplary thicknesses are providedprimarily for purposes of illustration, and that the disclosure is notlimited in these respects.

Thus it is to be understood that the chemFET arrays described herein maybe used to detect various analytes and, by doing so, may monitor avariety of reactions and/or interactions. It is also to be understoodthat the emphasis on hydrogen ion detection (in the form of a pH change)is for the sake of convenience and brevity and that other analytes(including other ions) can be substituted for hydrogen in thesedescriptions.

The chemFETs, including ISFETs, described herein are capable ofdetecting any analyte that is itself capable of inducing a change inelectric field. The analyte need not be charged in order to be detectedby the sensor. For example, depending on the embodiment, the analyte maybe positively charged (i.e., a cation), negatively charged (i.e., ananion), zwitterionic (i.e., capable of having two equal and oppositecharges but overall neutral), and polar yet neutral. This list is notintended as exhaustive as other analyte classes as well as specieswithin each class will be readily contemplated by those of ordinaryskill in the art based on the disclosure provided herein.

In the broadest sense of the invention, passivation layer may or may notbe coated and the analyte may or may not interact with the passivationlayer. As an example, the passivation layer may be comprised of siliconnitride and the analyte may be something other than hydrogen ions. As aspecific example, the passivation layer may be comprised of siliconnitride and the analyte may be inorganic pyrophosphate (PPi). In theseinstances, PPi is detected directly (i.e., in the absence of PPireceptors attached to the passivation layer either directly orindirectly).

If the analyte being detected is hydrogen (or alternatively hydroxide),then it is preferable to use weak buffers so that changes in eitherionic species can be detected at the passivation layer. If the analytebeing detected is something other than hydrogen (or hydroxide) but thereis some possibility of a pH change in the solution during the reactionor detection step, then it is preferable to use a strong buffer so thatchanges in pH do not interfere with the detection of the analyte. Abuffer is an ionic molecule that resists changes in pH. Buffers are ableto neutralize acids or bases added to or generated in a solution,resulting in no effective pH change in the solution. It is to beunderstood that any buffer is suitable provided it has a pKa in thedesired range. A suitable buffer is one that functions in about the pHrange of 6 to 9, and more preferably 6.5 to 8.5. The strength of abuffer is a relative term since it depends on the nature, strength andconcentration of the acid or base added to or generated in the solutionof interest. A weak buffer is a buffer that allows detection (andtherefore is not able to otherwise control) changes of about at least+/−0.005, 0.01, 0.015, 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20, 0.25,0.30, 0.35, 0.45, 0.50, or more. In some embodiments, the pH change ison the order of about 0.005 (e.g., per nucleotide incorporation) and ispreferably an increase in pH. A strong buffer is a buffer that controlspH changes of about at least +/−0.005, 0.01, 0.015, 0.02, 0.03, 0.04,0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.45, 0.50, or more. Bufferstrength can be varied by varying the concentration of the bufferspecies itself. Thus low concentration buffers can be low strengthbuffers. Examples include those having less than 1 mM (e.g., 50-100 μM)buffer species. A non-limiting example of a weak buffer suitable for thesequencing reactions described herein wherein change is the readout is0.1 mM Tris or Tricine. Examples of suitable weak buffers are providedin the Examples and are also known in the art. Higher concentrationbuffers can be stronger buffers. Examples include those having 1-25 mMbuffer species. A non-limiting example of a strong buffer suitable forthe sequencing reactions described herein wherein PPi is read directlyis 1, 5 or 25 mM (or more) Tris or Tricine. One of ordinary skill in theart will be able to determine the optimal buffer for use in thereactions and detection methods encompassed by the invention.

In some embodiments, the passivation layer and/or the molecules coatedis thereon dictate the analyte specificity of the array readout.

Detection of hydrogen ions (in the form of pH) can be carried out usinga passivation layer made of silicon nitride (Si₃N₄), silicon oxynitride(Si₂N₂O), silicon oxide (SiO₂), aluminum oxide (Al₂O₃), tantalumpentoxide (Ta₂O₅), tin oxide or stannic oxide (SnO₂), and the like.

The passivation layer can also detect other ion species directlyincluding but not limited to calcium, potassium, sodium, iodide,magnesium, chloride, lithium, lead, silver, cadmium, nitrate, phosphate,dihydrogen phosphate, and the like.

In some embodiments, the passivation layer is coated with a receptor forthe analyte of interest. The receptor binds selectively to the analyteof interest. As used herein, a receptor that binds selectively to ananalyte is a molecule that binds preferentially to that analyte (i.e.,its binding affinity for that analyte is greater than its bindingaffinity for any other analyte). Its binding affinity for the analyte ofinterest may be 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold,100-fold or more than its binding affinity for any other analyte. Inaddition to its relative binding affinity, the receptor must also havean absolute binding affinity that is sufficiently high to efficientlybind the analyte of interest (i.e., it must have a sufficientsensitivity). Receptors having binding affinities in the picomolar tomicromolar range are suitable. Preferably such interaction arereversible.

The receptor may be of any nature (e.g., chemical, nucleic acid,peptide, lipid, combinations thereof and the like). The analyte too maybe of any nature provided there exists a receptor that binds to itselectively and in some instances specifically. It is to be understoodhowever that the invention further contemplates detection of analytes inthe absence of a receptor. An example of this is the detection of PPiand Pi by the passivation layer in the absence of PPi or Pi receptors.

In one aspect, the invention contemplates receptors that are ionophores.As used herein, an ionophore is a molecule that binds selectively to anionic species, whether anion or cation. In the context of the invention,the ionophore is the receptor and the ion to which it binds is theanalyte. Ionophores of the invention include art-recognized carrierionophores (i.e., small lipid-soluble molecules that bind to aparticular ion) derived from microorganisms. Various ionophores arecommercially available from sources such as Calbiochem.

Detection of some ions can be accomplished through using the passivationlayer itself or through the use of receptors coated onto the passivationlayer. For example, potassium can be detected selectively using thepolysiloxane, valinomycin, or salinomycin; sodium can be detectedselectively using monensin, nystatin, or SQI-Pr; calcium can be detectedselectively using ionomycin, calcimycine (A23187), or CA 1001(ETI-11001).

Receptors able to bind more than one ion can also be used in someinstances. For example, beauvericin can be used to detect calcium and/orbarium ions, nigericin can be used to detect potassium, hydrogen and/orlead ions, and gramicidin can be used to detect hydrogen, sodium and/orpotassium ions. One of ordinary skill in the art will recognize thatthese compounds can be used in applications in which single ionspecificity is not required or in which it is unlikely (or impossible)that other ions which the compounds bind will be present or generated.

In other embodiments, including but not limited to nucleic acidsequencing applications, receptors that bind selectively to inorganicpyrophosphate (PPi) can be used. Examples of PPi receptors include thosecompounds shown in FIG. 11B (compounds 1-10). Compound 1 is described inAngew Chem Int Ed 2004 43:4777-4780 and US 2005/0119497 A 1 and isreferred to as p-naphthyl-bis[(bis(2-pyridylmethyl)amino)methyl]phenol.Compound 2 is described in J Am Chem Soc 2003 125:7752-7753 and US2005/0119497 A1 and is referred to asp-(p-nitrophenylazo)-bis[(bis(2-pyridylmethyl-1)amino)methyl]phenol (orits dinuclear Zn complex). Compound 3 is described in Sensors andActuators B 1995 29:324-327. Compound 4 is described in Angew Chem IntEd 2002 41(20):3811-3814. Compound 5 is described in WO 2007/002204 andis referred to therein as bis-Zn²⁺-dipicolylamine (Zn²⁺-DPA). Synthesisschemes for compounds 1 and 2 are shown provided in US 2005/0119497 A1.An exemplary synthesis for compound 4 is shown in FIG. 11C.

As another example, receptors for neurotoxins are described in SimonianElectroanalysis 2004, 16: 1896-1906.

Receptors may be attached to the passivation layer covalently ornon-covalently. Covalent attachment of a receptor to the passivationlayer may be direct or indirect (e.g., through a linker). FIG. 11Dillustrates the use of silanol chemistry to covalently bind receptors tothe passivation layer. Receptors may be immobilized on the passivationlayer using for example aliphatic primary amines (bottom left panel) oraryl isothiocyanates (bottom right panel). In these and otherembodiments, the passivation layer which itself may be comprised ofsilicon nitride, aluminum oxide, silicon oxide, tantalum pentoxide, orthe like, is bonded to a silanation layer via its reactive surfacegroups. For greater detail on silanol chemistry for covalent attachmentto the FET surface, reference can be made to at least the followingpublications: for silicon nitride, see Sensors and Actuators B 199529:324-327, Jpn J Appl Phys 1999 38:3912-3917 and Langmuir 200521:395-402; for silicon oxide, see Protein Sci 1995 4:2532-2544 and AmBiotechnol Lab 2002 20(7):16-18; and for aluminum oxide, see Colloidsand Surfaces 1992 63:1-9, Sensors and Accuators B 2003 89:40-47, andBioconjugate Chem 1997 8:424-433. The receptor is then conjugated to thesilanation layer reactive groups. This latter binding can occur directlyor indirectly through the use of a bifunctional linker, as illustratedin FIG. 11D. A bifunctional linker is a compound having at least tworeactive groups to which two entities may be bound. In some instances,the reactive groups are located at opposite ends of the linker. In someembodiments, the bifunctional linker is a universal bifunctional linkersuch as that shown in FIG. 11D. A universal linker is a linker that canbe used to link a variety of entities. It should be understood that thechemistries shown in FIG. 11D are meant to be illustrative and notlimiting.

The bifunctional linker may be a homo-bifunctional linker or ahetero-bifunctional linker, depending upon the nature of the moleculesto be conjugated. Homo-bifunctional linkers have two identical reactivegroups. Hetero-bifunctional linkers are have two different reactivegroups. Various types of commercially available linkers are reactivewith one or more of the following groups: primary amines, secondaryamines, sulphydryls, carboxyls, carbonyls and carbohydrates. Examples ofamine-specific linkers are bis(sulfosuccinimidyl)suberate,bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate,disuccinimidyl tartarate, dimethyl adipimate.2 HCl, dimethylpimelimidate.2 HCl, dimethyl suberimidate.2 HCl, and ethyleneglycolbis-[succinimidyl-[succinate]]. Linkers reactive with sulfhydrylgroups include bismaleimidohexane,1,4-di-[3′-(2′-pyridyldithio)-propionamido)]butane,1-[p-azidosalicylamido]-4-[iodoacetamido]butane, andN-[4-(p-azidosalicylamido)butyl]-3′-[2′-pyridyldithio]propionamide.Linkers preferentially reactive with carbohydrates include azidobenzoylhydrazine. Linkers preferentially reactive with carboxyl groups include4-[p-azidosalicylamido]butylamine.

Heterobifunctional linkers that react with amines and sulfhydrylsinclude N-succinimidyl-3-[2-pyridyldithio]propionate,succinimidyl[4-iodoacetyl]aminobenzoate, succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl6-[3-[2-pyridyldithio]propionamido]hexanoate, and sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate. Heterobifunctionallinkers that react with carboxyl and amine groups include1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride.Heterobifunctional linkers that react with carbohydrates and sulfhydrylsinclude 4-[N-maleimidomethyl]-cyclohexane-1-carboxylhydrazide.2 HCl,4-(4-N-maleimidophenyl)-butyric acid hydrazide.2 HCl, and3-[2-pyridyldithio]propionyl hydrazide.

Alternatively, receptors may be non-covalently coated onto thepassivation layer. Non-covalent deposition of the receptor to thepassivation layer may involve the use of a polymer matrix. The polymermay be naturally occurring or non-naturally occurring and may be of anytype including but not limited to nucleic acid (e.g., DNA, RNA, PNA,LNA, and the like, or mimics, derivatives, or combinations thereof),amino acid (e.g., peptides, proteins (native or denatured), and thelike, or mimics, derivatives, or combinations thereof, lipids,polysaccharides, and functionalized block copolymers. The receptor maybe adsorbed onto and/or entrapped within the polymer matrix.

Alternatively, the receptor may be covalently conjugated or crosslinkedto the polymer (e.g., it may be “grafted” onto a functionalizedpolymer).

An example of a suitable peptide polymer is poly-lysine (e.g.,poly-L-lysine). Examples of other polymers include block copolymers thatcomprise polyethylene glycol (PEG), polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitrocelluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, polyvinylchloride, polystyrene, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate), poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, polyanhydrides,poly(styrene-b-isobutylene-b-styrene) (SIBS) block copolymer, ethylenevinyl acetate, poly(meth)acrylic acid, polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone),and natural polymers such as alginate and other polysaccharidesincluding dextran and cellulose, collagen, albumin and other hydrophilicproteins, zein and other prolamines and hydrophobic proteins, copolymersand mixtures thereof, and chemical derivatives thereof includingsubstitutions and/or additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art.

Another issue that relates to ISFET threshold voltage stability and/orpredictability involves trapped charge that may accumulate on metallayers of CMOS-fabricated devices as a result of various processingactivities during or following array fabrication (e.g., back-end-of-lineprocessing such as plasma metal etching, wafer cleaning, dicing,packaging, handling, etc.). In particular, with reference to FIG. 11A,trapped charge may in some instances accumulate on one or more of thevarious conductors 304, 306, 308, 312, 316, 320, 326, 338, and 164constituting the ISFETs floating gate structure 170. This phenomenonalso is referred to in the relevant literature as the “antenna effect.”

One opportunity for trapped charge to accumulate includes plasma etchingof the topmost metal layer 304. Applicants have recognized andappreciated that other opportunities for charge to accumulate on one ormore conductors of the floating gate structure includes wafer dicing,during which the abrasive process of a dicing saw cutting through awafer generates static electricity, and/or various post-processing waferhandling/packaging steps, where automated machinery thathandles/transports wafers may be sources of electrostatic discharge(ESD) to conductors of the floating gate structure. If there is noconnection to the silicon substrate (or other semi-conductor substrate)to provide an electrical path to bleed off such charge accumulation,charge may build up to the point of causing undesirable changes ordamage to the gate oxide 165 (e.g., charge injection into the oxide, orlow-level oxide breakdown to the underlying substrate). Trapped chargein the gate oxide or at the gate oxide-semiconductor interface in turncan cause undesirable and/or unpredictable variations in ISFET operationand performance.

In view of the foregoing, other inventive embodiments of the presentdisclosure are directed to methods and apparatus for improving ISFETperformance by reducing trapped charge or mitigating the antenna effect.In one embodiment, trapped charge may be reduced after a sensor arrayhas been fabricated, while in other embodiments the fabrication processitself may be modified to reduce trapped charge that could be induced bysome conventional process steps. In yet other embodiments, both “duringfabrication” and “post fabrication” techniques may be employed incombination to reduce trapped charge and thereby improve ISFETperformance.

With respect to alterations to the fabrication process itself to reducetrapped charge, in one embodiment the thickness of the gate oxide 165shown in FIG. 11A may be particularly selected so as to facilitatebleeding of accumulated charge to the substrate; in particular, athinner gate oxide may allow a sufficient amount of built-up charge topass through the gate oxide to the substrate below without becomingtrapped. In another embodiment based on this concept, a pixel may bedesigned to include an additional “sacrificial” device, i.e., anothertransistor having a thinner gate oxide than the gate oxide 165 of theISFET. The floating gate structure of the ISFET may then be coupled tothe gate of the sacrificial device such that it serves as a “chargebleed-off transistor.” Of course, it should be appreciated that sometrade-offs for including such a sacrificial device include an increasein pixel size and complexity.

In another embodiment, the topmost metal layer 304 of the ISFETsfloating gate structure 170 shown in FIG. 11A may be capped with adielectric prior to plasma etching to mitigate trapped charge. Asdiscussed above, charge accumulated on the floating gate structure mayin some cases be coupled from the plasma being used for metal etching.Typically, a photoresist is applied over the metal to be etched and thenpatterned based on the desired geometry for the underlying metal. In oneexemplary implementation, a capping dielectric layer (e.g., an oxide)may be deposited over the metal to be etched, prior to the applicationof the photoresist, to provide an additional barrier on the metalsurface against charge from the plasma etching process. In one aspect,the capping dielectric layer may remain behind and form a portion of thepassivation layer 172.

In yet another embodiment, the metal etch process for the topmost metallayer 304 may be modified to include wet chemistry or ion-beam millingrather than plasma etching. For example, the metal layer 304 could beetched using an aqueous chemistry selective to the underlying dielectric(e.g., see http://www.transene.com/aluminum.html, hereby incorporatedherein by reference). Another alternative approach employs ion-millingrather than plasma etching for the metal layer 304. Ion-milling iscommonly used to etch materials that cannot be readily removed usingconventional plasma or wet chemistries. The ion-milling process does notemploy an oscillating electric field as does a plasma, so that chargebuild-up does not occur in the metal layer(s). Yet another metal etchalternative involves optimizing the plasma conditions so as to reducethe etch rate (i.e. less power density).

In yet another embodiment, architecture changes may be made to the metallayer to facilitate complete electrical isolation during definition ofthe floating gate. In one aspect, designing the metal stack-up so thatthe large area ISFET floating gate is not connected to anything duringits final definition may require a subsequent metal layer serving as a“jumper” to realize the electrical connection to the floating gate ofthe transistor. This “jumper” connection scheme prevents charge flowfrom the large floating gate to the transistor. This method may beimplemented as follows (M=metal layer): i) M1 contacting Poly gateelectrode; ii) M2 contacting M1; iii) M3 defines floating gate andseparately connects to M2 with isolated island; iv) M4 jumper, havingvery small area being etched over the isolated islands and connectionsto floating gate M3, connects the M3 floating gate to the M1/M2/M3 stackconnected to the Poly gate immediately over the transistor active area;and v) M3 to M4 interlayer dielectric is removed only over the floatinggate so as to expose the bare M3 floating gate. In the method outlinedimmediately above, step v) need not be done, as the ISFET architectureaccording to some embodiments discussed above leaves the M4 passivationin place over the M4 floating gate. In one aspect, removal maynonetheless improve ISFET performance in other ways (i.e. sensitivity).In any case, the final chemically-sensitive passivation layer may be athin sputter-deposited ion-sensitive metal-oxide layer. It should beappreciated that the over-layer jumpered architecture discussed abovemay be implemented in the standard CMOS fabrication flow to allow any ofthe first three metal layers to be used as the floating gates (i.e. M1,M2 or M3).

With respect to post-fabrication processes to reduce trapped charge, inone embodiment a “forming gas anneal” may be employed as apost-fabrication process to mitigate potentially adverse effects oftrapped charge. In a forming gas anneal, CMOS-fabricated ISFET devicesare heated in a hydrogen and nitrogen gas mixture. The hydrogen gas inthe mixture diffuses into the gate oxide 165 and neutralizes certainforms of trapped charges. In one aspect, the forming gas anneal need notnecessarily remove all gate oxide damage that may result from trappedcharges; rather, in some cases, a partial neutralization of some trappedcharge is sufficient to significantly improve ISFET performance. Inexemplary annealing processes according to the present disclosure,ISFETs may be heated for approximately 30 to 60 minutes at approximately400 to 425 degrees Celsius in a hydrogen/nitrogen mixture that includes10% to 15% hydrogen. In one particular implementation, annealing at 425degrees Celsius at 30 minutes in a hydrogen/nitrogen mixture thatincludes 10% hydrogen is observed to be particularly effective atimproving ISFET performance. For aluminum CMOS processes, thetemperature of the anneal should be kept at or below 450 degrees Celsiusto avoid damaging the aluminum metallurgy. In another aspect of anannealing process according to the present disclosure, the forming gasanneal is performed after wafers of fabricated ISFET arrays are diced,so as to ensure that damage due to trapped charge induced by the dicingprocess itself, and/or other pre-dicing processing steps (e.g., plasmaetching of metals) may be effectively ameliorated.

In yet other processes for mitigating potentially adverse effects oftrapped charge according to embodiments of the present disclosure, avariety of “electrostatic discharge {ESD)-sensitive protocols” may beadopted during any of a variety of wafer post-fabricationhandling/packaging steps. For example, in one exemplary process,anti-static dicing tape may be employed to hold wafer substrates inplace (e.g., during the dicing process). Also, although high-resistivity(e.g., 10 MΩ) deionized water conventionally is employed in connectionwith cooling of dicing saws, according to one embodiment of the presentdisclosure less resistive/more conductive water may be employed for thispurpose to facilitate charge conduction via the water; for example,deionized water may be treated with carbon dioxide to lower resistivityand improve conduction of charge arising from the dicing process.Furthermore, conductive and grounded die-ejection tools may be usedduring various wafer dicing/handling/packaging steps, again to provideeffective conduction paths for charge generated during any of thesesteps, and thereby reduce opportunities for charge to accumulate on oneor more conductors of the floating gate structure of respective ISFETsof an array.

In yet another embodiment involving a post-fabrication process to reducetrapped charge, the gate oxide region of an ISFET may be irradiated withUV radiation. With reference again to FIG. 11A, in one exemplaryimplementation based on this embodiment, an optional hole or window 302is included during fabrication of an ISFET array in the top metal layer304 of each pixel of the array, proximate to the ISFET floating gatestructure. This window is intended to allow UV radiation, whengenerated, to enter the ISFETs gate region; in particular, the variouslayers of the pixel 105 ₁. as shown in FIGS. 11 and 12 A-L, areconfigured such that UV radiation entering the window 302 may impinge inan essentially unobstructed manner upon the area proximate to thepolysilicon gate 164 and the gate oxide 165.

To facilitate a UV irradiation process to reduce trapped charge,Applicants have recognized and appreciated that materials other thansilicon nitride and silicon oxynitride generally need to be employed inthe passivation layer 172 shown in FIG. 11A, as silicon nitride andsilicon oxynitride significantly absorb UV radiation. In view of theforegoing, these materials need to be substituted with others that areappreciably transparent to UV radiation, examples of which include, butare not limited to, phososilicate glass (PSG) and boron-dopedphososilicate glass (BPSG). PSG and BPSG, however, are not impervious tohydrogen and hydroxyl ions; accordingly, to be employed in a passivationlayer of an ISFET designed for pH sensitivity, PSG and BPSG may be usedtogether with an ion-impervious material that is also significantlytransparent to UV radiation, such as aluminum oxide (Al₂O₃), to form thepassivation layer. For example, with reference again to FIG. 11A, PSG orBPSG may be employed as a substitute for silicon nitride or siliconoxynitride in the first portion 172A of the passivation layer 172, and athin layer (e.g., 400 to 600 Angstroms) of aluminum oxide may beemployed in the second portion 172B of the passivation layer 172 (e.g.,the aluminum oxide may be deposited using a post-CMOS lift-offlithography process).

In another aspect of an embodiment involving UV irradiation, each ISFETof a sensor array must be appropriately biased during a UV irradiationprocess to facilitate reduction of trapped charge. In particular, highenergy photons from the UV irradiation, impinging upon the bulk siliconregion 160 in which the ISFET conducting channel is formed, createelectron-hole pairs which facilitate neutralization of trapped charge inthe gate oxide as current flows through the ISFETs conducting channel.To this end, an array controller, discussed further below in connectionwith FIG. 17, generates appropriate signals for biasing the ISFETs ofthe array during a UV irradiation process. In particular, with referenceagain to FIG. 9, each of the signals RowSel₁ through RowSel_(n) isgenerated so as to enable/select (i.e., turn on) all rows of the sensorarray at the same time and thereby couple all of the ISFETs of the arrayto respective controllable current sources 106 _(j) in each column. Withall pixels of each column simultaneously selected, the current from thecurrent source 106 _(j) of a given column is shared by all pixels of thecolumn. The column amplifiers 107A and 107B are disabled by removing thebias voltage VB4, and at the same time the output of the amplifier 107B,connected to the drain of each ISFET in a given column, is grounded viaa switch responsive to a control signal “UV”. Also, the common bodyvoltage V_(BODY) for all ISFETs of the array is coupled to electricalground (i.e., V_(BODY)=0 Volts) (as discussed above, during normaloperation of the array, the body bias voltage V_(BODY) is coupled to thehighest voltage potential available to the array, e.g., VDDA). In oneexemplary procedure, the bias voltage VB1 for all of the controllablecurrent sources 106 _(j) is set such that each pixel's ISFET conductsapproximately 1 μA of current. With the ISFET array thusly biased, thearray then is irradiated with a sufficient dose of UV radiation (e.g.,from an EPROM eraser generating approximately 20 milli Watts/cm² ofradiation at a distance of approximately one inch from the array forapproximately 1 hour). After irradiation, the array may be allowed torest and stabilize over several hours before use for measurements ofchemical properties such as ion concentration.

FIG. 13 illustrates a block diagram of an exemplary CMOS IC chipimplementation of an ISFET sensor array 100 based on the column andpixel designs discussed above in connection with FIGS. 9-12, accordingto one embodiment of the present disclosure. In one aspect of thisembodiment, the array 100 includes 512 columns 102 ₁ through 102 ₅₁₂with corresponding column bias/readout circuitry 110 ₁ through 110 ₅₁₂(one for each column, as shown in FIG. 9), wherein each column includes512 geometrically square pixels 105 ₁ through 105 ₅₁₂, each having asize of approximately 9 micrometers by 9 micrometers (i.e., the array is512 columns by 512 rows). In another aspect, the entire array (includingpixels together with associated row and column select circuitry andcolumn bias/readout circuitry) may be fabricated on a semiconductor dieas an application specific integrated circuit (ASIC) having dimensionsof approximately 7 millimeters by 7 millimeters. While an array of 512by 512 pixels is shown in the embodiment of FIG. 13, it should beappreciated that arrays may be implemented with different numbers ofrows and columns and different pixel sizes according to otherembodiments, as discussed further below in connection with FIGS. 19-23.

Also, as discussed above, it should be appreciated that arrays accordingto various embodiments of the present invention may be fabricatedaccording to conventional CMOS fabrications techniques, as well asmodified CMOS fabrication techniques (e.g., to facilitate realization ofvarious functional aspects of the chemFET arrays discussed herein, suchas additional deposition of passivation materials, process steps tomitigate trapped charge, etc.) and other semiconductor fabricationtechniques beyond those conventionally employed in CMOS fabrication.Additionally, various lithography techniques may be employed as part ofan array fabrication process. For example, in one exemplaryimplementation, a lithography technique may be employed in whichappropriately designed blocks are “stitched” together by overlapping theedges of a step and repeat lithography exposures on a wafer substrate byapproximately 0.2 micrometers. In a single exposure, the maximum diesize typically is approximately 21 millimeters by 21 millimeters. Byselectively exposing different blocks (sides, top & bottoms, core, etc.)very large chips can be defined on a wafer (up to a maximum, in theextreme, of one chip per wafer, commonly referred to as “wafer scaleintegration”).

In one aspect of the array 100 shown in FIG. 13, the first and last twocolumns 102 ₁, 102 ₂, 102 ₅₁₁ and 102 ₅₁₂, as well as the first twopixels 105 ₁ and 105 ₂ and the last two pixels 105 ₅₁₁ and 105 ₅₁₂ ofeach of the columns 102 ₃ through 102 ₅₁₀ (e.g., two rows and columns ofpixels around a perimeter of the array) may be configured as “reference”or “dummy” pixels 103. With reference to FIG. 11A, for the dummy pixelsof an array, the topmost metal layer 304 of each dummy pixel's ISFET(coupled ultimately to the ISFETs polysilicon gate 164) is tied to thesame metal layer of other dummy pixels and is made accessible as aterminal of the chip, which in turn may be coupled to a referencevoltage VREF. As discussed above in connection with FIG. 9, thereference voltage VREF also may be applied to the bias/readout circuitryof respective columns of the array. In some exemplary implementationsdiscussed further below, preliminary test/evaluation data may beacquired from the array based on applying the reference voltage VREF andselecting and reading out dummy pixels, and/or reading out columns basedon the direct application of VREF to respective column buffers (e.g.,via the CAL signal), to facilitate offset determination (e.g.,pixel-to-pixel and column-to-column variances) and array calibration.

In FIG. 13, various power supply and bias voltages required for arrayoperation (as discussed above in connection with FIG. 9) are provided tothe array via electrical connections (e.g., pins, metal pads) andlabeled for simplicity in block 195 as “supply and bias connections.”The array 100 of FIG. 13 also includes a row select shift register 192,two sets of column select shift registers 194 _(1,2) and two outputdrivers 198 ₁ and 198 ₂ to provide two parallel output signals from thearray, Vout1 and Vout2, representing sensor measurements. The variouspower supply and bias voltages, control signals for the row and columnshift registers, and control signals for the column bias/readoutcircuitry shown in FIG. 13 are provided by an array controller, asdiscussed further below in connection with FIG. 17, which also reads theoutput signals Vout1 and Vout2 (and other optional status/diagnosticsignals) from the array 100. In another aspect of the array embodimentshown in FIG. 13, configuring the array such that multiple regions(e.g., multiple columns) of the array may be read at the same time viamultiple parallel array outputs (e.g., Vout1 and Vout2) facilitatesincreased data acquisition rates, as discussed further below inconnection with FIGS. 17 and 18. While FIG. 13 illustrates an arrayhaving two column select registers and parallel output signals Vout1 andVout2 to acquire data simultaneously from two columns at a time, itshould be appreciated that, in other embodiments, arrays according tothe present disclosure may be configured to have only one measurementsignal output, or more than two measurement signal outputs; inparticular, as discussed further below in connection with FIGS. 19-23,more dense arrays according to other inventive embodiments may beconfigured to have four our more parallel measurement signal outputs andsimultaneously enable different regions of the array to provide data viathe four our more outputs.

FIG. 14 illustrates the row select shift register 192, FIG. 15illustrates one of the column select shift registers 194, and FIG. 16illustrates one of the output drivers 198, of the array 100 shown inFIG. 13, according to one exemplary implementation. As shown in FIGS. 14and 15, the row and column select shift registers are implemented as aseries of D-type flip-flops coupled to a digital circuitry positivesupply voltage VDDD and a digital supply ground VSSD. In the row andcolumn shift registers, a data signal is applied to a D-input of firstflip-flop in each series and a clock signal is applied simultaneously toa clock input of all of the flip-flops in the series. For eachflip-flop, a “Q” output reproduces the state of the D-input upon atransition (e.g., falling edge) of the clock signal. With reference toFIG. 14, the row select shift register 192 includes 512 D-typeflip-flops, in which a first flip-flop 193 receives a vertical datasignal DV and all flip-flops receive a vertical clock signal CV. A “Q”output of the first flip-flop 193 provides the first row select signalRowSel₁ and is coupled to the D-input of the next flip-flop in theseries. The Q outputs of successive flip-flops are coupled to theD-inputs of the next flip-flop in the series and provide the row selectsignals RowSel₂ through RowSel₅₁₂ with successive falling edgetransitions of the vertical clock signal CV, as discussed further belowin connection with FIG. 18. The last row select signal RowSel₅₁₂ alsomay be taken as an optional output of the array 100 as the signal LSTV(Last STage Vertical), which provides an indication (e.g., fordiagnostic purposes) that the last row of the array has been selected.While not shown explicitly in FIG. 14, each of the row select signalsRowSel₁ through RowSel₅₁₂ is applied to a corresponding inverter, theoutput of which is used to enable a given pixel in each column (asillustrated in FIG. 9 by the signals RowSel₁ through RowSel_(n) ).

Regarding the column select shift registers 194 ₁ and 194 ₂, these areimplemented in a manner similar to that of the row select shiftregisters, with each column select shift register comprising 256series-connected flip-flops and responsible for enabling readout fromeither the odd columns of the array or the even columns of the array.For example, FIG. 15 illustrates the column select shift register 194 ₂,which is configured to enable readout from all of the even numberedcolumns of the array in succession via the column select signalsColSel₂, ColSel₄, . . . COLSEL₅₁₂, whereas another column select shiftregister 194 ₁ is configured to enable readout from all of the oddnumbered columns of the array in succession (via column select signalsColSel₁, ColSel₃, . . . ColSel₅₁₁). Both column select shift registersare controlled simultaneously by the horizontal data signal DH and thehorizontal clock signal CH to provide the respective column selectsignals, as discussed further below in connection with FIG. 18. As shownin FIG. 15, the last column select signal ColSel₅₁₂ also may be taken asan optional output of the array 100 as the signal LSTH (Last STageHorizontal), which provides an indication (e.g., for diagnosticpurposes) that the last column of the array has been selected.

With reference again for the moment to FIG. 7, Applicants haverecognized and appreciated that an implementation for array row andcolumn selection based on shift registers, as discussed above inconnection with FIGS. 13-15, is a significant improvement to the row andcolumn decoder approach employed in various prior art ISFET arraydesigns, including the design of Milgrew et al. shown in FIG. 7. Inparticular, regarding the row decoder 92 and the column decoder 94 shownin FIG. 7, the complexity of implementing these components in anintegrated circuit array design increases dramatically as the size ofthe array is increased, as additional inputs to both decoders arerequired. For example, an array having 512 rows and columns as discussedabove in connection with FIG. 13 would require nine inputs (2⁹=512) perrow and column decoder if such a scheme were employed for row and columnselection; similarly, arrays having 7400 rows and 7400 columns, asdiscussed below in connection with other embodiments, would require 13inputs (2¹³=8192) per row and column decoder. In contrast, the row andcolumn select shift registers shown in FIGS. 14 and 15 require noadditional input signals as array size is increased, but ratheradditional D-type flip-flops (which are routinely implemented in a CMOSprocess). Thus, the shift register implementations shown in FIGS. 14 and15 provide an easily scalable solution to array row and columnselection.

In the embodiment of FIG. 13, the “odd” column select shift register 194₁ provides odd column select signals to an “odd” output driver 198 ₁ andthe even column select shift register 194 ₂ provides even column selectsignals to an “even” output driver 198 ₂. Both output drivers areconfigured similarly, and an example of the even output driver 198 ₂ isshown in FIG. 16. In particular, FIG. 16 shows that respective evencolumn output signals V_(COL2), V_(COL4), . . . V_(COL512) (refer toFIG. 9 for the generic column signal output V_(COLj)) are applied tocorresponding switches 191 ₂, 191 ₄, . . . 191 ₅₁₂, responsive to theeven column select signals ColSel₂, ColSel₄, . . . ColSel₅₁₂ provided bythe column select register 194 ₂, to successively couple the even columnoutput signals to the input of a buffer amplifier 199 (BUF) via a bus175. In FIG. 16, the buffer amplifier 199 receives power from an outputbuffer positive supply voltage VDDO and an output buffer supply groundVSSO, and is responsive to an output buffer bias voltage VBO0 to set acorresponding bias current for the buffer output. Given the highimpedance input of the buffer amplifier 199, a current sink 197responsive to a bias voltage VB3 is coupled to the bus 175 to provide anappropriate drive current (e.g., on the order of approximately 100 μA)for the output of the column output buffer (see the buffer amplifier 111j of FIG. 9) of a selected column. The buffer amplifier 199 provides theoutput signal Vout2 based on the selected even column of the array; atthe same time, with reference to FIG. 13, a corresponding bufferamplifier of the “odd” output driver 198 ₁ provides the output signalVout1 based on a selected odd column of the array.

In one exemplary implementation, the switches of both the even and oddoutput drivers 198 ₁ and 198 ₂ (e.g., the switches 191 ₂, 191 ₄, . . .191 ₅₁₂ shown in FIG. 16) may be implemented as CMOS-pair transmissiongates (including an n-channel MOSFET and a p-channel MOSFET; see FIG.4), and inverters may be employed so that each column select signal andits complement may be applied to a given transmission gate switch 191 toenable switching. Each switch 191 has a series resistance when enabledor “on” to couple a corresponding column output signal to the bus 175;likewise, each switch adds a capacitance to the bus 175 when the switchis off. A larger switch reduces series resistance and allows a higherdrive current for the bus 175, which generally allows the bus 175 tosettle more quickly; on the other hand, a larger switch increasescapacitance of the bus 175 when the switch is off, which in turnincreases the settling time of the bus 175. Hence, there is a trade-offbetween switch series resistance and capacitance in connection withswitch size.

The ability of the bus 175 to settle quickly following enabling ofsuccessive switches in turn facilitates rapid data acquisition from thearray. To this end, in some embodiments the switches 191 of the outputdrivers 198 ₁ and 198 ₂ are particularly configured to significantlyreduce the settling time of the bus 175. Both the n-channel and thep-channel MOSFETs of a given switch add to the capacitance of the bus175; however, n-channel MOSFETs generally have better frequency responseand current drive capabilities than their p-channel counterparts. Inview of the foregoing, Applicants have recognized and appreciated thatsome of the superior characteristics of n-channel MOSFETs may beexploited to improve settling time of the bus 175 by implementing“asymmetric” switches in which respective sizes for the n-channel MOSFETand p-channel MOSFET of a given switch are different.

For example, in one embodiment, with reference to FIG. 16, the currentsink 197 may be configured such that the bus 175 is normally “pulleddown” when all switches 191 ₂, 191 ₄, . . . 191 ₅₁₂ are open or off (notconducting). Given a somewhat limited expected signal dynamic range forthe column output signals based on ISFET measurements, when a givenswitch is enabled or on (conducting), in many instances most of theconduction is done by the n-channel MOSFET of the CMOS-pair constitutingthe switch. Accordingly, in one aspect of this embodiment, the n-channelMOSFET and the p-channel MOSFET of each switch 191 are sizeddifferently; namely, in one exemplary implementation, the n-channelMOSFET is sized to be significantly larger than the p-channel MOSFET.More specifically, considering equally-sized n-channel and p-channelMOSFETs as a point of reference, in one implementation the n-channelMOSFET may be increased to be about 2 to 2.5 times larger, and thep-channel MOSFET may be decreased in size to be about 8 to 10 timessmaller, such that the n-channel MOSFET is approximately 20 times largerthan the p-channel MOSFET. Due to the significant decrease is size ofthe p-channel MOSFET and the relatively modest increase in size of then-channel MOSFET, the overall capacitance of the switch in the off stateis notably reduced, and there is a corresponding notable reduction incapacitance for the bus 175; at the same time, due to the largern-channel MOSFET, there is a significant increase in current drivecapability, frequency response and transconductance of the switch, whichin turn results in a significant reduction in settling time of the bus175.

While the example above describes asymmetric switches 191 for the outputdrivers 198 ₁ and 198 ₂ in which the n-channel MOSFET is larger than thep-channel MOSFET, it should be appreciated that in another embodiment,the converse may be implemented, namely, asymmetric switches in whichthe p-channel MOSFET is larger than the n-channel MOSFET. In one aspectof this embodiment, with reference again to FIG. 16, the current sink197 may alternatively serve as a source of current to appropriatelydrive the output of the column output buffer (see the buffer amplifier111 j of FIG. 9) of a selected column, and be configured such that thebus 175 is normally “pulled up” when all switches 191 ₂, 191 ₄, . . .191 ₅₁₂ are open or off (not conducting). In this situation, most of theswitch conduction may be accomplished by the p-channel MOSFET of theCMOS-pair constituting the switch. Benefits of reduced switchcapacitance (and hence reduced bus capacitance) may be realized in thisembodiment, although the overall beneficial effect of reduced settlingtime for the bus 175 may be somewhat less than that described previouslyabove, due to the lower frequency response of p-channel MOSFETs ascompared to n-channel MOSFETs. Nevertheless, asymmetric switches basedon larger p-channel MOSFETs may still facilitate a notable reduction inbus settling time, and may also provide for circuit implementations inwhich the column output buffer amplifier (111 j of FIG. 9) may be abody-tied source follower with appreciably increased gain.

In yet another embodiment directed to facilitating rapid settling of thebus 175 shown in FIG. 16, it may be appreciated that fewer switches 191coupled to the bus 175 results in a smaller bus capacitance. With thisin mind, and with reference again to FIG. 13, in yet another embodiment,more than two output drivers 198 ₁ and 198 ₂ may be employed in theISFET array 100 such that each output driver handles a smaller number ofcolumns of the array. For example, rather than having all even columnshandled by one driver and all odd columns handled by another driver, thearray may include four column select registers 194 _(1,2,3,4) and fourcorresponding output drivers 198 _(1,2,3,4) such that each output driverhandles one-fourth of the total columns of the array, rather thanone-half of the columns. In such an implementation, each output driverwould accordingly have half the number of switches 191 as compared withthe embodiment discussed above in connection with FIG. 16, and the bus175 of each output driver would have a corresponding lower capacitance,thereby improving bus settling time. While four output drivers arediscussed for purposes of illustration in this example, it should beappreciated that the present disclosure is not limited in this respect,and virtually any number of output drivers greater than two may beemployed to improve bus settling time in the scenario described above.Other array embodiments in which more than two output drivers areemployed to facilitate rapid data acquisition from the array arediscussed in greater detail below (e.g., in connection with FIGS.19-23).

In one aspect of the array design discussed above in connection withFIGS. 13-16, separate analog supply voltage connections (for VDDA,VSSA), digital supply voltage connections (for VDDD, VSSD) and outputbuffer supply voltage connections (for VDDO,VSSO) are provided on thearray to facilitate noise isolation and reduce signal cross-talk amongstvarious array components, thereby increasing the signal-to-noise ratio(SNR) of the output signals Vout1 and Vout2. In one exemplaryimplementation, the positive supply voltages VDDA, VDDD and VDDO eachmay be approximately 3.3 Volts. In another aspect, these voltagesrespectively may be provided “off chip” by one or more programmablevoltage sources, as discussed further below in connection with FIG. 17.

FIG. 17 illustrates a block diagram of the sensor array 100 of FIG. 13coupled to an array controller 250, according to one inventiveembodiment of the present disclosure. In various exemplaryimplementations, the array controller 250 may be fabricated as a “standalone” controller, or as a computer compatible “card” forming part of acomputer 260, as discussed above in connection with FIG. 8. In oneaspect, the functions of the array controller 250 may be controlled bythe computer 260 through an interface block 252 (e.g., serial interface,via USB port or PCI bus, Ethernet connection, etc.), as shown in FIG.17. In one embodiment, the array controller 250 is fabricated as aprinted circuit board into which the array 100 plugs, similar to aconventional IC chip (e.g., the array 100 is configured as an ASIC thatplugs into the array controller). In one aspect of such an embodiment,all or portions of the array controller 250 may be implemented as afield programmable gate array (FPGA) configured to perform various arraycontroller functions described in further detail below.

Generally, the array controller 250 provides various supply voltages andbias voltages to the array 100, as well as various signals relating torow and column selection, sampling of pixel outputs and dataacquisition. In particular, the array controller 250 reads the twoanalog output signals Vout1 (odd columns) and Vout2 (even columns)including multiplexed respective pixel voltage signals from the array100 and then digitizes these respective pixel signals to providemeasurement data to the computer 260, which in turn may store and/orprocess the data. In some implementations, the array controller 250 alsomay be configured to perform or facilitate various array calibration anddiagnostic functions, and an optional array UV irradiation treatment asdiscussed above in connection with FIG. 11A.

As illustrated in FIG. 17, the array controller 250 generally providesto the array 100 the analog supply voltage and ground (VDDA, VSSA), thedigital supply is voltage and ground (VDDD, VSSD), and the buffer outputsupply voltage and ground (VDDO, VSSO). In one exemplary implementation,each of the supply voltages VDDA, VDDD and VDDO is approximately 3.3Volts. As discussed above, in one aspect each of these power supplyvoltages is provided to the array 100 via separate conducting paths tofacilitate noise isolation. In another aspect, these supply voltages mayoriginate from respective power supplies/regulators, or one or more ofthese supply voltages may originate from a common source in a powersupply 258 of the array controller 250. The power supply 258 also mayprovide the various bias voltages required for array operation (e.g.,VB1, VB2, VB3, VB4, VBO0, V_(BODY)) and the reference voltage VREF usedfor array diagnostics and calibration. In another aspect, the powersupply 258 includes one or more digital-to-analog converters (DACs) thatmay be controlled by the computer 260 to allow any or all of the biasvoltages, reference voltage, and supply voltages to be changed undersoftware control (i.e., programmable bias settings). For example, apower supply 258 responsive to computer control may facilitateadjustment of the bias voltages VB1 and VB2 for pixel drain current, VB3for column bus drive, VB4 for column amplifier bandwidth, and VBO0 forcolumn output buffer current drive. In some aspects, one or more biasvoltages may be adjusted to optimize settling times of signals fromenabled pixels. Additionally, the common body voltage V_(BODY) for allISFETs of the array may be grounded during an optional post-fabricationUV irradiation treatment to reduce trapped charge, and then coupled to ahigher voltage (e.g., VDDA) during diagnostic analysis, calibration, andnormal operation of the array for measurement/data acquisition.Likewise, the reference voltage VREF may be varied to facilitate avariety of diagnostic and calibration functions.

As also shown in FIG. 17, the reference electrode 76 which is typicallyemployed in connection with an analyte solution to be measured by thearray 100 (as discussed above in connection with FIG. 1), may be coupledto the power supply 258 to provide a reference potential for the pixeloutput voltages. For example, in one implementation the referenceelectrode 76 may be coupled to a supply ground (e.g., the analog groundVSSA) to provide a reference for the pixel output voltages based on Eq.(3) above. In other exemplary implementations, the reference electrodevoltage may be set by placing a solution/sample of interest having aknown pH level in proximity to the sensor array 100 and adjusting thereference electrode voltage until the array output signals Vout1 andVout2 provide pixel voltages at a desired reference level, from whichsubsequent changes in pixel voltages reflect local changes in pH withrespect to the known reference pH level. In general, it should beappreciated that a voltage associated with the reference electrode 76need not necessarily be identical to the reference voltage VREFdiscussed above (which may be employed for a variety of array diagnosticand calibration functions), although in some implementations thereference voltage VREF provided by the power supply 258 may be used toset the voltage of the reference electrode 76.

Regarding data acquisition from the array 100, in one embodiment thearray controller 250 of FIG. 17 may include one or more preamplifiers253 to further buffer the output signals Vout1 and Vout2 from the sensorarray and provide selectable gain. In one aspect, the array controller250 may include one preamplifier for each output signal (e.g., twopreamplifiers for two analog output signals). In other aspects, thepreamplifiers may be configured to accept input voltages from 0.0 to 3.3Volts, may have programmable/computer selectable gains (e.g., 1, 2, 5,10 and 20) and low noise outputs (e.g., <10 nV/sqrtHz), and may providelow pass filtering (e.g., bandwidths of 5 MHz and 25 MHz). In yetanother aspect, the preamplifiers may have a programmable/computerselectable offset for input and/or output voltage signals to set anominal level for either to a desired range.

The array controller 250 of FIG. 17 also comprises one or moreanalog-to-digital converters 254 (ADCs) to convert the sensor arrayoutput signals Vout1 and Vout2 to digital outputs (e.g., 10-bit or12-bit) so as to provide data to the computer 260. In one aspect, oneADC may be employed for each analog output of the sensor array, and eachADC may be coupled to the output of a corresponding preamplifier (ifpreamplifiers are employed in a given implementation). In anotheraspect, the ADC(s) may have a computer-selectable input range (e.g., 50mV, 200 mV, 500 mV, 1 V) to facilitate compatibility with differentranges of array output signals and/or preamplifier parameters. In yetother aspects, the bandwidth of the ADC(s) may be greater than 60 MHz,and the data acquisition/conversion rate greater than 25 MHz (e.g., ashigh as 100 MHz or greater).

In the embodiment of FIG. 17, ADC acquisition timing and array row andcolumn selection may be controlled by a timing generator 256. Inparticular, the timing generator provides the digital vertical data andclock signals (DV, CV) to control row selection, the digital horizontaldata and clock signals (DH, CH) to control column selection, and thecolumn sample and hold signal COL SH to sample respective pixel voltagesfor an enabled row, as discussed above in connection with FIG. 9. Insome implementations, the timing generator 256 may be implemented by amicroprocessor executing code and configured as a multi-channel digitalpattern generator to provide appropriately timed control signals. In oneexemplary implementation, the timing generator 256 may be implemented asa field-programmable gate array (FPGA).

FIG. 18 illustrates an exemplary timing diagram for such signals, asprovided by the timing generator 256, to acquire pixel data from thesensor array 100. For purposes of the following discussion, a “frame” isdefined as a data set that includes a value (e.g.; pixel output signalor voltage V_(S)) for each pixel in the array, and a “frame rate” isdefined as the rate at which successive frames may be acquired from thearray. In the example of FIG. 18, an exemplary frame rate of 20frames/sec is chosen to illustrate operation of the array (i.e., row andcolumn selection and signal acquisition); however, it should beappreciated that arrays and array controllers according to the presentdisclosure are not limited in this respect, as different frame rates,including lower frame rates (e.g., 1 to 10 frames/second) or higherframe rates (e.g., 25, 30, 40, 50, 60, 70 to 100 frames/sec., etc.),with arrays having the same or higher numbers of pixels, are possible.In some exemplary applications, a data set may be acquired that includesmany frames over several seconds to conduct an experiment on a givenanalyte or analytes. Several such experiments may be performed insuccession, in some cases with pauses in between to allow for datatransfer/processing and/or washing of the sensor array ASIC and reagentpreparation for a subsequent experiment.

In one implementation, the array controller 250 controls the array 100to enable rows successively, one at a time. For example, with referenceagain for the moment to FIG. 9, a first row of pixels is enabled via therow select signal RowSel₁ . The enabled pixels are allowed to settle forsome time period, after which the COL SH signal is asserted briefly toclose the sample/hold switch in each column and store on the column'ssample/hold capacitor Csh the voltage value output by the first pixel inthe column. This voltage is then available as the column output voltageV_(COLj) applied to one of the two (odd and even column) array outputdrivers 198 ₁ and 198 ₂ (e.g., see FIG. 16). The COL SH signal is thende-asserted, thereby opening the sample/hold switches in each column anddecoupling the column output buffer 111 j from the column amplifiers107A and 107B. Shortly thereafter, the second row of pixels is enabledvia the row select signal RowSel₂ . During the time period in which thesecond row of pixels is allowed to settle, the column select signals aregenerated two at a time (one odd and one even; odd column select signalsare applied in succession to the odd output driver, even column selectsignals are applied in succession to the even output driver) to read thecolumn output voltages associated with the first row. Thus, while agiven row in the array is enabled and settling, the previous row isbeing read out, two columns at a time. By staggering row selection andsampling/readout, and by reading multiple columns at a time for a givenrow, a frame of data may be acquired from the array in a significantlystreamlined manner.

FIG. 18 illustrates the timing details of the foregoing process for anexemplary frame rate of 20 frames/sec. Given this frame rate and 512rows in the array, each row must be read out in approximately 98microseconds, as indicated by the vertical delineations in FIG. 18.Accordingly, the vertical clock signal CV has a period of 98microseconds (i.e., a clock frequency of over 10 kHz), with a new rowbeing enabled on a trailing edge (negative transition) of the CV signal.The left side of FIG. 18 reflects the beginning of a new frame cycle, atwhich point the vertical data signal DV is asserted before a firsttrailing edge of the CV signal and de-asserted before the next trailingedge of the CV signal (for data acquisition from successive frames, thevertical data signal is reasserted again only after row 512 is enabled).Also, immediately before each trailing edge of the CV signal (i.e., newrow enabled), the COL SH signal is asserted for 2 microseconds, leavingapproximately 50 nanoseconds before the trailing edge of the CV signal.

In FIG. 18, the first occurrence of the COL SH signal is actuallysampling the pixel values of row 512 of the array. Thus, upon the firsttrailing edge of the CV signal, the first row is enabled and allowed tosettle (for approximately 96 microseconds) until the second occurrenceof the COL SH signal. During this settling time for the first row, thepixel values of row 512 are read out via the column select signals.Because two column select signals are generated simultaneously to read512 columns, the horizontal clock signal CH must generate 256 cycleswithin this period, each trailing edge of the CH signal generating oneodd and one even column select signal. As shown in FIG. 18, the firsttrailing edge of the CH signal in a given row is timed to occur twomicroseconds after the selection of the row to allow for settling of thevoltage values stored on the sample/hold capacitors C_(Sh) and providedby the column output buffers. Also for each row, the horizontal datasignal DH is asserted before the first trailing edge of the CH signaland de-asserted before the next trailing edge of the CH signal. The lasttwo columns (e.g., 511 and 512) are selected before the occurrence ofthe COL SH signal which, as discussed above, occurs approximately twomicroseconds before the next row is enabled. Thus, 512 columns are read,two at a time, within a time period of approximately 94 microseconds(i.e., 98 microseconds per row, minus two microseconds at the beginningand end of each row). This results in a data rate for each of the arrayoutput signals Vout1 and Vout2 of approximately 2.7 MHz. In anotheraspect, the ADC(s) 254 may be controlled by the timing generator 256 tosample the output signals Vout1 and Vout2 at a significantly higher rateto provide multiple digitized samples for each pixel measurement, whichmay then be averaged (e.g., the ADC data acquisition rate may beapproximately 100 MHz to sample the 2.7 MHz array output signals,thereby providing as many as approximately 35-40 samples per pixelmeasurement).

In addition to controlling the sensor array and ADCs, the timinggenerator 256 may be configured to facilitate various array calibrationand diagnostic functions, as well as an optional UV irradiationtreatment. To this end, the timing generator may utilize the signal LSTVindicating the selection of the last row of the array and the signalLSTH to indicate the selection of the last column of the array. Thetiming generator 256 also may be responsible for generating the CALsignal which applies the reference voltage VREF to the column bufferamplifiers, and generating the UV signal which grounds the drains of allISFETs in the array during a UV irradiation process (see FIG. 9). Thetiming is generator also may provide some control function over thepower supply 258 during various calibration and diagnostic functions, orUV irradiation, to appropriately control supply or bias voltages; forexample, during UV irradiation, the timing generator may control thepower supply to couple the body voltage V_(BODY) to ground while the UVsignal is activated to ground the ISFET drains. With respect to arraycalibration and diagnostics, as well as UV irradiation, in someimplementations the timing generator may receive specialized programsfrom the computer 260 to provide appropriate control signals. In oneaspect, the computer 260 may use various data obtained from dummy pixelsof the array, as well as column information based on the application ofthe CAL signal and the reference voltage VREF, to determine variouscalibration parameters associated with a given array and/or generatespecialized programs for calibration and diagnostic functions.

With respect to the computer interface 252 of the array controller 250,in one exemplary implementation the interface is configured tofacilitate a data rate of approximately 200 MB/sec to the computer 260,and may include local storage of up to 400 MB or greater. The computer260 is configured to accept data at a rate of 200 MB/sec, and processthe data so as to reconstruct an image of the pixels (e.g., which may bedisplayed in false-color on a monitor). For example, the computer may beconfigured to execute a general-purpose program with routines written inC++ or Visual Basic to manipulate the data and display is as desired.

Having discussed several aspects of an exemplary ISFET array and anarray controller according to the present disclosure, FIGS. 19-23illustrate block diagrams of alternative CMOS IC chip implementations ofISFET sensor arrays having greater numbers of pixels, according to yetother inventive embodiments. In one aspect, each of the ISFET arraysdiscussed further below in connection with FIGS. 19-23 may be controlledby an array controller similar to that shown in FIG. 17, in some caseswith minor modifications to accommodate higher numbers of pixels (e.g.,additional preamplifiers 253 and analog-to-digital converters 254).

FIG. 19 illustrates a block diagram of an ISFET sensor array 100A basedon the column and pixel designs discussed above in connection with FIGS.9-12 and a 0.35 micrometer CMOS fabrication process, according to oneinventive embodiment. The array 100A includes 2048 columns 102 ₁ through101 ₂₀₄₈, wherein each column includes 2048 geometrically square pixels105 ₁ through 105 ₂₀₄₈, each having a size of approximately 9micrometers by 9 micrometers. Thus, the array includes over four millionpixels (>4 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 20.5millimeters by 20.5 millimeters.

In one aspect of the embodiment shown in FIG. 19, the array 100A may beconfigured, at least in part, as multiple groups of pixels that may berespectively controlled. For example, each column of pixels may bedivided into top and bottom halves, and the collection of pixels inrespective top halves of columns form a first group 4001 of rows (e.g.,a top group, rows 1-1024) and the collection of pixels in respectivebottom halves of columns form a second group 400 ₂ of rows (e.g., abottom group, rows 1025-2048). In turn, each of the first and second(e.g., top and bottom) groups of rows is associated with correspondingrow select registers, column bias/readout circuitry, column selectregisters, and output drivers. In this manner, pixel selection and dataacquisition from each of the first and second groups of rows 400 ₁ and400 ₂ is substantially similar to pixel selection and data acquisitionfrom the entire array 100 shown in FIG. 13; stated differently, in oneaspect, the array 100A of FIG. 19 substantially comprises twosimultaneously controlled “sub-arrays” of different pixel groups toprovide for significantly streamlined data acquisition from highernumbers of pixels.

In particular, FIG. 19 shows that row selection of the first group 400 ₁of rows may be controlled by a first row select register 192 ₁, and rowselection of the second group 400 ₂ of rows may be controlled by asecond row select register 192 ₂. In one aspect, each of the row selectregisters 192 ₁ and 192 ₂ may be configured as discussed above inconnection with FIG. 14 to receive vertical clock (CV) and vertical data(DV) signals and generate row select signals in response; for examplethe first row select register 192 ₁ may generate the signals RowSel₁through RowSel₁₀₂₄ and the second row select register 192 ₂ may generatethe signals RowSel₁₀₂₅ through RowSel₂₀₄₈ . In another aspect, both rowselect registers 192 ₁ and 192 ₂ may simultaneously receive commonvertical clock and data signals, such that two rows of the array areenabled at any given time, one from the top group and another from thebottom group.

For each of the first and second groups of rows, the array 100A of FIG.19 further comprises column bias/readout circuitry 110 _(1T)-110_(2048T) (for the first row group 400 ₁) and 110 _(1B)-110 _(2048B) (forthe second row group 400 ₂), such that each column includes twoinstances of the bias/readout circuitry 110 j shown in FIG. 9. The array100A also comprises two column select registers 192 _(1,2) (odd andeven) and two output drivers 198 _(1,2) (odd and even) for the secondrow group 400 ₂, and two column select registers 192 _(3,4) (odd andeven) and two output drivers 198 _(3,4) (odd and even) for the first rowgroup 400 ₁ (i.e., a total of four column select registers and fouroutput drivers). The column select registers receive horizontal clocksignals (CHT and CHB for the first row group and second row group,respectively) and horizontal data signals (DHT and DHB for the first rowgroup and second row group, respectively) to control odd and even columnselection. In one implementation, the CHT and CHB signals may beprovided as common signals, and the DHT and DHB may be provided ascommon signals, to simultaneously read out four columns at a time fromthe array (i.e., one odd and one even column from each row group); inparticular, as discussed above in connection with FIGS. 13-18, twocolumns may be simultaneously read for each enabled row and thecorresponding pixel voltages provided as two output signals. Thus, viathe enablement of two rows at any given time, and reading of two columnsper row at any given time, the array 100A may provide four simultaneousoutput signals Vout1, Vout2, Vout3 and Vout4.

In one exemplary implementation of the array 100A of FIG. 19, in whichcomplete data frames (all pixels from both the first and second rowgroups 400 ₁ and 400 ₂) are acquired at a frame rate of 20 frames/sec,1024 pairs of rows are successively enabled for periods of approximately49 microseconds each. For each enabled row, 1024 pixels are read out byeach column select register/output driver during approximately 45 tomicroseconds (allowing 2 microseconds at the beginning and end of eachrow, as discussed above in connection with FIG. 18). Thus, in thisexample, each of the array output signals Vout1, Vout2, Vout3 and Vout4has a data rate of approximately 23 MHz. Again, it should be appreciatedthat in other implementations, data may be acquired from the array 100Aof FIG. 19 at frame rates other than 20 frames/sec (e.g., 50-100frames/sec).

Like the array 100 of FIG. 13, in yet other aspects the array 100A ofFIG. 19 may include multiple rows and columns of dummy or referencepixels 103, around a perimeter of the array to facilitate preliminarytest/evaluation data, offset determination an/or array calibration.Additionally, various power supply and bias voltages required for arrayoperation (as discussed above in connection with FIG. 9) are provided tothe array 100A in block 195, in a manner similar to that discussed abovein connection with FIG. 13.

FIG. 20 illustrates a block diagram of an ISFET sensor array 100B basedon a 0.35 micrometer CMOS fabrication process and having a configurationsubstantially similar to the array 100A discussed above in FIG. 19,according to yet another inventive embodiment. While the array 100B alsois based generally on the column and pixel designs discussed above inconnection with FIGS. 9-12, the pixel size/pitch in the array 100B issmaller than that of the pixel shown in FIG. 10. In particular, withreference again to FIGS. 10 and 11, the dimension “e” shown in FIG. 10is substantially reduced in the embodiment of FIG. 20, without affectingthe integrity of the active pixel components disposed in the centralregion of the pixel, from approximately 9 micrometers to approximately 5micrometers; similarly, the dimension “f” shown in FIG. 10 is reducedfrom approximately 7 micrometers to approximately 4 micrometers. Stateddifferently, some of the peripheral area of the pixel surrounding theactive components is substantially reduced with respect to thedimensions given in connection with FIG. 10, without disturbing thetop-view and cross-sectional layout and design of the pixel's activecomponents as shown in FIGS. 10 and 11. A top view of such a pixel 105Ais shown in FIG. 20A, in which the dimension “e” is 5.1 micrometers andthe dimension “f” is 4.1 micrometers. In one aspect of this pixeldesign, to facilitate size reduction, fewer body connections B areincluded in the pixel 105A (e.g., one at each corner of the pixel) ascompared to the pixel shown in FIG. 10, which includes several bodyconnections B around the entire perimeter of the pixel.

As noted in FIG. 20, the array 100B includes 1348 columns 102 ₁ through102 ₁₃₄₈, wherein each column includes 1152 geometrically square pixels105A₁ through 105A₁₁₅₂, each having a size of approximately 5micrometers by 5 micrometers. Thus, the array includes over 1.5 millionpixels (>1.5 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 9millimeters by 9 millimeters. Like the array 100A of FIG. 19, in oneaspect the array 100B of FIG. 20 is divided into two groups of rows 400₁ and 400 ₂, as discussed above in connection with FIG. 19. In oneexemplary implementation, complete data frames (all pixels from both thefirst and second row groups 400 ₁ and 400 ₂) are acquired at a framerate of 50 frames/sec, thereby requiring 576 pairs of rows to besuccessively enabled for periods of approximately 35 microseconds each.For each enabled row, 674 pixels are read out by each column selectregister/output driver during approximately 31 microseconds (allowing 2microseconds at the beginning and end of each row, as discussed above inconnection with FIG. 18). Thus, in this example, each of the arrayoutput signals Vout1, Vout2, Vout3 and Vout4 has a data rate ofapproximately 22 MHz. Again, it should be appreciated that in otherimplementations, data may be acquired from the array 100B of FIG. 20 atframe rates other than 50 frames/sec.

FIG. 21 illustrates a block diagram of an ISFET sensor array 100C basedon a 0.35 micrometer CMOS fabrication process and incorporating thesmaller pixel size discussed above in connection with FIGS. 20 and 20A(5.1 micrometer square pixels), according to yet another embodiment. Asnoted in FIG. 21, the array 100C includes 4000 columns 102, through 102₄₀₀₀, wherein each column includes 3600 geometrically square pixels105A₁ through 105A₃₆₀₀, each having a size of approximately 5micrometers by 5 micrometers. Thus, the array includes over 14 millionpixels (>14 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 22millimeters by 22 millimeters. Like the arrays 100A and 100B of FIGS. 19and 20, in one aspect the array 100C of FIG. 21 is divided into twogroups of rows 400 ₁ and 400 ₂. However, unlike the arrays 100A and100B, for each row group the array 100C includes sixteen column selectregisters and sixteen output drivers to simultaneously read sixteenpixels at a time in an enabled row, such that thirty-two output signalsVout1-Vout32 may be provided from the array 100C. In one exemplaryimplementation, complete data frames (all pixels from both the first andsecond row groups 400 ₁ and 400 ₂) may be acquired at a frame rate of 50frames/sec, thereby requiring 1800 pairs of rows to be successivelyenabled for periods of approximately 11 microseconds each. For eachenabled row, 250 pixels (4000/16) are read out by each column selectregister/output driver during approximately 7 microseconds (allowing 2microseconds at the beginning and end of each row). Thus, in thisexample, each of the array output signals Vout1-Vout32 has a data rateof approximately 35 MHz. As with the previous embodiments, it should beappreciated that in other implementations, data may be acquired from thearray 100C at frame rates other than 50 frames/sec.

While the exemplary arrays discussed above in connection with FIGS.13-21 are based on a 0.35 micrometer conventional CMOS fabricationprocess, it should be appreciated that arrays according to the presentdisclosure are not limited in this respect, as CMOS fabricationprocesses having feature sizes of less than 0.35 micrometers may beemployed (e.g., 0.18 micrometer CMOS processing techniques) to fabricatesuch arrays. Accordingly, ISFET sensor arrays with a pixel size/pitchsignificantly below 5 micrometers may be fabricated, providing forsignificantly denser ISFET arrays. For example, FIGS. 22 and 23illustrate respective block diagrams of ISFET sensor arrays 100D and100E according to yet other embodiments based on a 0.18 micrometer CMOSfabrication process, in which a pixel size of 2.6 micrometers isachieved. The pixel design itself is based substantially on the pixel105A shown in FIG. 20A, albeit on a smaller scale due to the 0.18micrometer CMOS process.

The array 100D of FIG. 22 includes 2800 columns 102 ₁ through 102 ₂₈₀₀,wherein each column includes 2400 geometrically square pixels eachhaving a size of approximately 2.6 micrometers by 2.6 micrometers. Thus,the array includes over 6.5 million pixels (>6.5 Mega-pixels) and, inone exemplary implementation, the complete array (ISFET pixels andassociated circuitry) may be fabricated as an integrated circuit chiphaving dimensions of approximately 9 millimeters by 9 millimeters. Likethe arrays 100A, 100B and 100C of FIGS. 19-21, in one aspect the array100D of FIG. 22 is divided into two groups of rows 400 ₁ and 400 ₂.However, unlike the arrays 100A, 100B, and 100C, for each row group thearray 100D includes eight column select registers and eight outputdrivers to simultaneously read eight pixels at a time in an enabled row,such that sixteen output signals Vout1-Vout16 may be provided from thearray 100D. In one exemplary implementation, complete data frames (allpixels from both the first and second row groups 400 ₁ and 400 ₂) may beacquired at a frame rate of 50 frames/sec, thereby requiring 1200 pairsof rows to be successively enabled for periods of approximately 16-17microseconds each. For each enabled row, 350 pixels (2800/8) are readout by each column select register/output driver during approximately 14microseconds (allowing 1 to 2 microseconds at the beginning and end ofeach row). Thus, in this example, each of the array output signalsVout1-Vout16 has a data rate of approximately 25 MHz. As with theprevious embodiments, it should be appreciated that in otherimplementations, data may be acquired from the array 100D at frame ratesother than 50 frames/sec.

The array 100E of FIG. 23 includes 7400 columns 102 ₁ through 102 ₇₄₀₀,wherein each column includes 7400 geometrically square pixels eachhaving a size of approximately 2.6 micrometers by 2.6 micrometers. Thus,the array includes over 54 million pixels (>54 Mega-pixels) and, in oneexemplary implementation, the complete array (ISFET pixels andassociated circuitry) may be fabricated as an integrated circuit chiphaving dimensions of approximately 21 millimeters by 21 millimeters.Like the arrays 100A-100D of FIGS. 19-22, in one aspect the array 100Eof FIG. 23 is divided into two groups of rows 400 ₁ and 400 ₂. However,unlike the arrays 100A-100D, for each row group the array 100E includesthirty-two column select registers and thirty-two output drivers tosimultaneously read thirty-two pixels at a time in an enabled row, suchthat sixty-four output signals Vout1-Vout64 may be provided from thearray 100E. In one exemplary implementation, complete data frames (allpixels from both the first and second row groups 400 ₁ and 400 ₂) may beacquired at a frame rate of 100 frames/sec, thereby requiring 3700 pairsof rows to be successively enabled for periods of approximately 3microseconds each. For each enabled row, 230 pixels (7400/32) are readout by each column select register/output driver during approximately700 nanoseconds. Thus, in this example, each of the array output signalsVout1-Vout64 has a data rate of approximately 328 MHz. As with theprevious embodiments, it should be appreciated that in otherimplementations, data may be acquired from the array 100D at frame ratesother than 100 frames/sec.

Thus, in various examples of ISFET arrays based on the inventiveconcepts disclosed herein, an array pitch of approximately nine (9)micrometers (e.g., a sensor surface area of less than ten micrometers byten micrometers) allows an ISFET array including over 256,000 pixels(i.e., a 512 by 512 array), together with associated row and columnselect and bias/readout electronics, to be fabricated on a 7 millimeterby 7 millimeter semiconductor die, and a similar sensor array includingover four million pixels (i.e., a 2048 by 2048 array, over 4Mega-pixels) to be fabricated on a 21 millimeter by 21 millimeter die.In other examples, an array pitch of approximately 5 micrometers allowsan ISFET array including approximately 1.55 Mega-pixels (i.e., a 1348 by1152 array) and associated electronics to be fabricated on a 9millimeter by 9 millimeter die, and an ISFET sensor array including over14 Mega-pixels and associated electronics on a 22 millimeter by 20millimeter die. In yet other implementations, using a CMOS fabricationprocess in which feature sizes of less than 0.35 micrometers arepossible (e.g., 0.18 micrometer CMOS processing techniques), ISFETsensor arrays with a pixel size/pitch significantly below 5 micrometersmay be fabricated (e.g., array pitch of 2.6 micrometers or pixel/sensorarea of less than 8 or 9 micrometers²), providing for significantlydense ISFET arrays.

In the embodiments of ISFET arrays discussed above, array pixels employa p-channel ISFET, as discussed above in connection with FIG. 9. Itshould be appreciated, however, that ISFET arrays according to thepresent disclosure are not limited in this respect, and that in otherembodiments pixel designs for ISFET arrays may be based on an n-channelISFET. In particular, any of the arrays discussed above in connectionwith FIGS. 13 and 19-23 may be implemented with pixels based onn-channel ISFETs.

For example, FIG. 24 illustrates the pixel design of FIG. 9 implementedwith an n-channel ISFET and accompanying n-channel MOSFETs, according toanother inventive embodiment of the present disclosure. Morespecifically, FIG. 24 illustrates one exemplary pixel 105 ₁ of an arraycolumn (i.e., the first pixel of the column), together with columnbias/readout circuitry 110 j, in which the ISFET 150 (Q1) is ann-channel ISFET. Like the pixel design of FIG. 9, the pixel design ofFIG. 24 includes only three components, namely, the ISFET 150 and twon-channel MOSFET switches Q2 and Q3, responsive to one of n row selectsignals (RowSel₁ through RowSel_(n), logic high active). No transmissiongates are required in the pixel of FIG. 24, and all devices of the pixelare of a “same type,” i.e., n-channel devices. Also like the pixeldesign of FIG. 9, only four signal lines per pixel, namely the lines 112₁, 114 ₁, 116 ₁ and 118 ₁ are required to operate the three componentsof the pixel 105 ₁ shown in FIG. 24. In other respects, the pixeldesigns of FIG. 9 and FIG. 24 are similar, in that they are bothconfigured with a constant drain current I_(Dj) and a constantdrain-to-source voltage V_(DSj) to obtain an output signal V_(Sj) froman enabled pixel.

One of the primary differences between the n-channel ISFET pixel designof FIG. 24 and the p-channel ISFET design of FIG. 9 is the oppositedirection of current flow through the pixel. To this end, in FIG. 24,the element 106 _(j) is a controllable current sink coupled to theanalog circuitry supply voltage ground VSSA, and the element 108 _(j) ofthe bias/readout circuitry 110 _(j) is a controllable current sourcecoupled to the analog positive supply voltage VDDA. Additionally, thebody connection of the ISFET 150 is not tied to its source, but ratherto the body connections of other ISFETs of the array, which in turn iscoupled to the analog ground VSSA, as indicated in FIG. 24.

In addition to the pixel designs shown in FIGS. 9 and 24 (based on aconstant ISFET drain current and constant ISFET drain-source voltage),alternative pixel designs are contemplated for ISFET arrays, based onboth p-channel ISFETs and n-channel ISFETs, according to yet otherinventive embodiments of the present disclosure, as illustrated in FIGS.25-27. As discussed below, some alternative pixel designs may requireadditional and/or modified signals from the array controller 250 tofacilitate data acquisition. In particular, a common feature of thepixel designs shown in FIGS. 25-27 includes a sample and hold capacitorwithin each pixel itself, in addition to a sample and hold capacitor foreach column of the array. While the alternative pixel designs of FIGS.25-27 generally include a greater number of components than the pixeldesigns of FIGS. 9 and 24, the feature of a pixel sample and holdcapacitor enables “snapshot” types of arrays, in which all pixels of anarray may be enabled simultaneously to sample a complete frame andacquire signals representing measurements of one or more analytes inproximity to respective ISFETs of the array. In some applications, thismay provide for higher data acquisition speeds and/or improved signalsensitivity (e.g., higher signal-to-noise ratio).

FIG. 25 illustrates one such alternative design for a single pixel 105Cand associated column circuitry 110 j. The pixel 105C employs ann-channel ISFET and is based generally on the premise of providing aconstant voltage across the ISFET Q1 based on a feedback amplifier (Q4,Q5 and Q6). In particular, transistor Q4 constitutes the feedbackamplifier load, and the amplifier current is set by the bias voltage VB1(provided by the array controller). Transistor Q5 is a common gateamplifier and transistor Q6 is a common source amplifier. Again, thepurpose of feedback amplifier is to hold the voltage across the ISFET Q1constant by adjusting the current supplied by transistor Q3. TransistorQ2 limits the maximum current the ISFET Q1 can draw (e.g., so as toprevent damage from overheating a very large array of pixels). Thismaximum current is set by the bias voltage VB2 (also provided by thearray controller). In one aspect of the pixel design shown in FIG. 25,power to the pixel 105C may be turned off by setting the bias voltageVB2 to 0 Volts and the bias voltage VB1 to 3.3 Volts. In this manner,the power supplied to large arrays of such pixels may be modulated(turned on for a short time period and then off by the array controller)to obtain ion concentration measurements while at the same time reducingoverall power consumption of the array. Modulating power to the pixelsalso reduces heat dissipation of the array and potential heating of theanalyte solution, thereby also reducing any potentially deleteriouseffects from sample heating.

In FIG. 25, the output of the feedback amplifier (the gate of transistorQ3) is sampled by MOS switch Q7 and stored on a pixel sample and holdcapacitor Csh within the pixel itself. The switch Q7 is controlled by apixel sample and hold signal pSH (provided to the array chip by thearray controller), which is applied simultaneously to all pixels of thearray so as to simultaneously store the readings of all the pixels ontheir respective sample and hold capacitors. In this manner, arraysbased on the pixel design of FIG. 25 may be considered as “snapshot”arrays, in that a full frame of data is sampled at any given time,rather than sampling successive rows of the array. After each pixelvalue is stored on the corresponding pixel sample and hold capacitorCsh, each pixel 105C (ISFET and feedback amplifier) is free to acquireanother pH reading or it can by turned off to conserve power.

In FIG. 25, the pixel values stored on all of the pixel sample and holdcapacitors Csh are applied to the column circuitry 110 j one row at atime through source follower Q8, which is enabled via the transistor Q9in response to a row select signal (e.g., RowSel1). In particular, aftera row is selected and has settled out, the values stored in the pixelsample and hold capacitors are then in turn stored on the column sampleand hold capacitors Csh2, as enabled by the column sample and holdsignal COL SH, and provided as the column output signal V_(COLj).

FIG. 26 illustrates another alternative design for a single pixel 105Dand associated column circuitry 110 j, according to one embodiment ofthe present disclosure. In this embodiment, the ISFET is shown as ap-channel device. At the start of a data acquisition cycle, CMOSswitches controlled by the signals pSH (pixel sample/hold) and pRST(pixel reset) are closed (these signals are supplied by the arraycontroller). This pulls the source of ISFET (Q1) to the voltage VRST.Subsequently, the switch controlled by the signal pRST is opened, andthe source of ISFET Q1 pulls the pixel sample and hold capacitor Csh toa threshold below the level set by pH. The switch controlled by thesignal pSH is then opened, and the pixel output value is coupled, viaoperation of a switch responsive to the row select signal RowSel1, tothe column circuitry 110 j to provide the column output signal V_(COLj).Like the pixel design in the embodiment illustrated in FIG. 25, arraysbased on the pixel 105D are “snapshot” type arrays in that all pixels ofthe array may be operated simultaneously. In one aspect, this designallows a long simultaneous integration time on all pixels followed by ahigh-speed read out of an entire frame of data.

FIG. 27 illustrates yet another alternative design for a single pixel105E and associated column circuitry 110 j, according to one embodimentof the present disclosure. In this embodiment, again the ISFET is shownas a p-channel device. At the start of a data acquisition cycle, theswitches operated by the control signals p1 and pRST are briefly closed.This clears the value stored on the sampling capacitor Csh and allows acharge to be stored on ISFET (Q1). Subsequently, the switch controlledby the signal pSH is closed, allowing the charge stored on the ISFET Q1to be stored on the pixel sample and hold capacitor Csh. The switchcontrolled by the signal pSH is then opened, and the pixel output valueis coupled, via operation of a switch responsive to the row selectsignal RowSel1, to the column circuitry 110 j to provide the columnoutput signal V_(COLj). Gain may be provided in the pixel 105E via theratio of the ISFET capacitance to the Csh cap, i.e., gain=C_(Q1)/C_(sh),or by enabling the pixel multiple times (i.e., taking multiple samplesof the analyte measurement) and accumulating the ISFET output on thepixel sample and hold capacitor Csh without resetting the capacitor(i.e., gain is a function of the number of accumulations). Like theembodiments of FIGS. 25 and 26, arrays based on the pixel 105D are“snapshot” type arrays in that all pixels of the array may be operatedsimultaneously.

Turning from the sensor discussion, we will now be addressing thecombining of the ISFET array with a microwell array and the attendantfluidics. As most of the drawings of the microwell array structure arepresented only in cross-section or showing that array as only a block ina simplified diagram, FIGS. 28A and 28B are provided to assist thereader in beginning to visualize the resulting apparatus inthree-dimensions. FIG. 28A shows a group of round cylindrical wells 2810arranged in an array, while FIG. 28B shows a group of rectangularcylindrical wells 2830 arranged in an array. It will be seen that thewells are separated (isolated) from each other by the material 2840forming the well walls. While it is certain possible to fabricate wellsof other cross sections, it is not believed to be advantageous to do so.Such an array of microwells sits over the above-discussed ISFET array,with one or more ISFETs per well. In the subsequent drawings, when themicrowell array is identified, one may picture one of these arrays.

Fluidic System: Apparatus and Method for Use with High DensityElectronic Sensor Arrays

For many uses, to complete a system for sensing chemical reactions orchemical agents using the above-explained high density electronicarrays, techniques and apparatus are required for delivery to the arrayelements (called “pixels”) fluids containing chemical or biochemicalcomponents for sensing. In this section, exemplary techniques andmethods will be illustrated, which are useful for such purposes, withdesirable characteristics.

As high speed operation of the system may be desired, it is preferredthat the fluid delivery system, insofar as possible, not limit the speedof operation of the overall system.

Accordingly, needs exist not only for high-speed, high-density arrays ofISFETs or other elements sensitive to ion concentrations or otherchemical attributes, or changes in chemical attributes, but also forrelated mechanisms and techniques for supplying to the array elementsthe samples to be evaluated, in sufficiently small reaction volumes asto substantially advance the speed and quality of detection of thevariable to be sensed.

There are two and sometimes three components or subsystems, and relatedmethods, involved in delivery of the subject chemical samples to thearray elements: (1) macrofluidic system of reagent and wash fluidsupplies and appropriate valving and ancillary apparatus, (2) a flowcell and (3) in many applications, a microwell array. Each of thesesubsystems will be discussed, though in reverse order.

Microwell Array

As discussed elsewhere, for many uses, such as in DNA sequencing, it isdesirable to provide over the array of semiconductor sensors acorresponding array of microwells, each microwell being small enoughpreferably to receive only one DNA-loaded bead, in connection with whichan underlying pixel in the array will provide a corresponding outputsignal.

The use of such a microwell array involves three stages of fabricationand preparation, each of which is discussed separately: (1) creating thearray of microwells to result in a chip having a coat comprising amicrowell array layer; (2) mounting of the coated chip to a fluidicinterface; and in the case of DNA sequencing, (3) loading DNA-loadedbead or beads into the wells. It will be understood, of course, that inother applications, beads may be unnecessary or beads having differentcharacteristics may be employed.

Microwell Array Fabrication

Microwell fabrication may be accomplished in a number of ways. Theactual details of fabrication may require some experimentation and varywith the processing capabilities that are available.

In general, fabrication of a high density array of microwells involvesphoto-lithographically patterning the well array configuration on alayer or layers of material such as photoresist (organic or inorganic),a dielectric, using an etching process. The patterning may be done withthe material on the sensor array or it may be done separately and thentransferred onto the sensor array chip, of some combination of the two.However, techniques other than photolithography are not to be excludedif they provide acceptable results.

One example of a method for forming a microwell array is now discussed,starting with reference to FIG. 29. That figure diagrammatically depictsa top view of one corner (i.e., the lower left corner) of the layout ofa chip showing an array 2910 of the individual ISFET sensors 2912 on theCMOS die 2914. Signal lines 2916 and 2918 are used for addressing thearray and reading its output. Block 2920 represents some of theelectronics for the array, as discussed above, and layer 2922 representsa portion of a wall which becomes part of a microfluidics structure, theflow cell, as more fully explained below; the flow cell is thatstructure which provides a fluid flow over the microwell array or overthe sensor surface directly, if there is no microwell structure. On thesurface of the die, a pattern such as pattern 2922 at the bottom left ofFIG. 29 may be formed during the semiconductor processing to form theISFETs and associated circuitry, for use as alignment marks for locatingthe wells over the sensor pixels when the dielectric has covered thedie's surface.

After the semiconductor structures, as shown, are formed, the microwellstructure is applied to the die. That is, the microwell structure can beformed right on the die or it may be formed separately and then mountedonto the die, either approach being acceptable. To form the microwellstructure on the die, various processes may be used. For example, theentire die may be spin-coated with, for example, a negative photoresistsuch as Microchem's SU-8 2015 or a positive resist/polyimide such as HDMicrosystems HD8820, to the desired height of the microwells. Thedesired height of the wells (e.g., about 4-12 μm in the example of onepixel per well, though not so limited as a general matter) in thephotoresist layer(s) can be achieved by spinning the appropriate resistat predetermined rates (which can be found by reference to theliterature and manufacturer specifications, or empirically), in one ormore layers. (Well height typically may be selected in correspondencewith the lateral dimension of the sensor pixel, preferably for a nominal1:1-1.5:1 aspect ratio, height:width or diameter. Based onsignal-to-noise considerations, there is a relationship betweendimensions and the required data sampling rates to achieve a desiredlevel of performance. Thus there are a number of factors that will gointo selecting optimum parameters for a given application.)Alternatively, multiple layers of different photoresists may be appliedor another form of dielectric material may be deposited. Various typesof chemical vapor deposition may also be used to build up a layer ofmaterials suitable for microwell formation therein.

Once the photoresist layer (the singular form “layer” is used toencompass multiple layers in the aggregate, as well) is in place, theindividual wells (typically mapped to have either one or four ISFETsensors per well) may be generated by placing a mask (e.g., of chromium)over the resist-coated die and exposing the resist to cross-linking(typically UV) radiation. All resist exposed to the radiation (i.e.,where the mask does not block the radiation) becomes cross-linked and asa result will form a permanent plastic layer bonded to the surface ofthe chip (die). Unreacted resist (i.e., resist in areas which are notexposed, due to the mask blocking the light from reaching the resist andpreventing cross-linking) is removed by washing the chip in a suitablesolvent (i.e., developer) such as propyleneglycolmethylethylacetate(PGMEA) or other appropriate solvent. The resultant structure definesthe walls of the microwell array.

FIG. 30 shows an example of a layout for a portion of a chromium mask3010 for a one-sensor-per-well embodiment, corresponding to the portionof the die shown in FIG. 29. The grayed areas 3012, 3014 are those thatblock the UV radiation. The alignment marks in the white portions 3016on the bottom left quadrant of FIG. 30, within gray area 3012, are usedto align the layout of the wells with the ISFET sensors on the chipsurface. The array of circles 3014 in the upper right quadrant of themask block radiation from reaching the well areas, to leave unreactedresist which can be dissolved in forming the wells.

FIG. 31 shows a corresponding layout for the mask 3020 for a4-sensors-per-well embodiment. Note that the alignment pattern 3016 isstill used and that the individual well-masking circles 3014A in thearray 2910 now have twice the diameter as the wells 3014 in FIG. 30, foraccommodating four sensors per well instead of one sensor-per-well.

After exposure of the die/resist to the UV radiation, a second layer ofresist may be coated on the surface of the chip. This layer of resistmay be relatively thick, such as about 400-450 μm thick, typically. Asecond mask 3210 (FIG. 32), which also may be of chromium, is used tomask an area 3220 which surrounds the array, to build a collar or wall(or basin, using that term in the geological sense) 3310 of resist whichsurrounds the active array of sensors on substrate 3312, as shown inFIG. 33. In the particular example being described, the collar is 150 μmwider than the sensor array, on each side of the array, in the xdirection, and 9 μm wider on each side than the sensor array, in the ydirection. Alignment marks on mask 3210 (most not shown) are matched upwith the alignment marks on the first layer and the CMOS chip itself.

Other photolithographic approaches may be used for formation of themicrowell array, of course, the foregoing being only one example.

For example, contact lithography of various resolutions and with variousetchants and developers may be employed. Both organic and inorganicmaterials may be used for the layer(s) in which the microwells areformed. The layer(s) may be etched on a chip having a dielectric layerover the pixel structures in the sensor array, such as a passivationlayer, or the layer(s) may be formed separately and then applied overthe sensor array. The specific choice or processes will depend onfactors such as array size, well size, the fabrication facility that isavailable, acceptable costs, and the like.

Among the various organic materials which may be used in someembodiments to form the microwell layer(s) are the above-mentioned SU-8type of negative-acting photoresist, a conventional positive-actingphotoresist and a positive-acting photodefineable polyimide. Each hasits virtues and its drawbacks, well known to those familiar with thephotolithographic art.

Naturally, in a production environment, modifications will beappropriate.

Contact lithography has its limitations and it may not be the productionmethod of choice to produce the highest densities of wells—i.e., it mayimpose a higher than desired minimum pitch limit in the lateraldirections. Other techniques, such as a deep UV step-and-repeat process,are capable of providing higher resolution lithography and can be usedto produce small pitches and possibly smaller well diameters. Of course,for different desired specifications (e.g., numbers of sensors and wellsper chip), different techniques may prove optimal. And pragmaticfactors, such as the fabrication processes available to a manufacturer,may motivate the use of a specific fabrication method. While novelmethods are discussed, various aspects of the invention are limited touse of these novel methods.

Preferably the CMOS wafer with the ISFET array will be planarized afterthe final metallization process. A chemical mechanical dielectricplanarization prior to the silicon nitride passivation is suitable. Thiswill allow subsequent lithographic steps to be done on very flatsurfaces which are free of back-end CMOS topography.

By utilizing deep-UV step-and-repeat lithography systems, it is possibleto resolve small features with superior resolution, registration, andrepeatability. However, the high resolution and large numerical aperture(NA) of these systems precludes their having a large depth of focus. Assuch, it may be necessary, when using such a fabrication system, to usethinner photodefinable spin-on layers (i.e., resists on the order of 1-2μm rather than the thicker layers used in contact lithography) topattern transfer and then etch microwell features to underlying layer orlayers. For example, four 1 μm plasma-enhanced chemical vapor thin-filmdepositions (standard fab process) may be done sequentially to render atarget microwell thickness of 4 μm. High resolution lithography can thenbe used to pattern the microwell features and conventional SiO2 etchchemistries can be used—one each for the bondpad areas and then themicrowell areas—having selective etch stops; the etch stops then can beon aluminum bondpads and silicon nitride passivation (or the like),respectively. Alternatively, other suitable substitute pattern transferand etch processes can be employed to render microwells of inorganicmaterials.

Another approach is to form the microwell structure in an organicmaterial. For example, a dual-resist “soft-mask” process may beemployed, whereby a thin high-resolution deep-UV resist is used on topof a thicker organic material (e.g., cured polyimide or opposite-actingresist). The top resist layer is patterned. The pattern can betransferred using an oxygen plasma reactive ion etch process. Thisprocess sequence is sometimes referred to as the “portable conformablemask” (PCM) technique. See B. J. Lin et al., “Practicing the Novolacdeep-UV portable conformable masking technique”, Journal of VacuumScience and Technology 19, No. 4, 1313-1319 (1981); and A. Cooper et al,“Optimization of a photosensitive spin-on dielectric process for copperinductor coil and interconnect protection in RF SoC devices.”

Alternatively a “drill-focusing” technique may be employed, wherebyseveral sequential step-and-repeat exposures are done at different focaldepths to compensate for the limited depth of focus (DOF) ofhigh-resolution steppers when patterning thick resist layers. Thistechnique depends on the stepper NA and DOF as well as the contrastproperties of the resist material.

Another PCM technique may be adapted to these purposes, such as thatshown in U.S. patent application publication no. 2006/0073422 by Edwardset al. This is a three-layer PCM process and it is illustrated in FIG.33A. As shown there, basically six major steps are required to producethe microwell array and the result is quite similar to what contactlithography would yield.

In a first step, 3320, a layer of high contrast negative-actingphotoresist such as type Shipley InterVia Photodielectric Material 8021(IV8021) 3322 is spun on the surface of a wafer, which we shall assumeto be the wafer providing the substrate 3312 of FIG. 33 (in which thesensor array is fabricated), and a soft bake operation is performed.Next, in step 3324, a blocking anti-reflective coating (BARC) layer3326, is applied and soft baked. On top of this structure, a thin resistlayer 3328 is spun on and soft baked, step 3330, the thin layer ofresist being suitable for fine feature definition. The resist layer 3328is then patterned, exposed and developed, and the BARC in the exposedregions 3329, not protected any longer by the resist 3328, is removed,Step 3332. This opens up regions 3329 down to the uncured IV8021 layer.The BARC layer can now act like a conformal contact mask. A blanketexposure with a flooding exposure tool, Step 3334, cross-links theexposed IV8021, which is now shown as distinct from the uncured IV8021at 3322. One or more developer steps 3338 are then performed, removingeverything but the cross-linked IV8021 in regions 3336. Regions 3336 nowconstitute the walls of the microwells.

Although as shown above, the wells bottom out (i.e. terminate) on thetop passivation layer of the ISFETs, it is believed that an improvementin ISFET sensor performance (i.e. such as signal-to-noise ratio) can beobtained if the active bead(s) is(are) kept slightly elevated from theISFET passivation layer. One way to do so is to place a spacer “bump”within the boundary of the pixel microwell. An example of how this couldbe rendered would be not etching away a portion of the layer-or-layersused to form the microwell structure (i.e. two lithographic steps toform the microwells—one to etch part way done, the other to pattern thebump and finish the etch to bottom out), by depositing andlithographically defining and etching a separate layer to form the“bump”, or by using a permanent photo-definable material for the bumponce the microwells are complete. The bump feature is shown as 3350 inFIG. 33B. An alternative (or additional) non-integrated approach is toload the wells with a layer or two of very small packing beads beforeloading the DNA-bearing beads.

Mounting the Coated Chip to a Flow Cell (Fluidic Interface)

The process of using the assembly of an array of sensors on a chipcombined with an array of microwells to sequence the DNA in a sample isreferred to as an “experiment.” Executing an experiment requires loadingthe wells with the DNA-bound beads and the flowing of several differentsolutions (i.e., reagents and washes) across the wells. A fluid deliverysystem coupled with a fluidic interface is needed which flows thevarious solutions across the wells in a controlled laminar flow withacceptably small dead volumes and small cross contamination betweensequential solutions. The fluidic interface is sometimes referred to asa “flow cell.”

Flow cell designs of many configurations are possible; the system andmethods presented herein are not dependent on use of a specific flowcell configuration. It is desirable, though, that a suitable flow cellsubstantially conform to the following set of objectives:

-   -   suitable interconnections with a fluidics delivery system—e.g.,        via appropriately-sized tubing;    -   appropriate (for dna sequencing, approximately 300 μm) head        space above wells;    -   minimization of dead volumes encountered by fluids prior to        their entry to the flow chamber (i.e., the enclosed space above        the microwell array).    -   elimination of small spaces in contact with liquid but not swept        by through the flow cell (to minimize cross contamination).    -   uniform expansion of flow from the inlet tubing to a broad/flat        front at the entry to the flow chamber.    -   laminar flow characteristics such that the broad/flat front        profile is maintained as it traverses across the chip from inlet        side to outlet side.    -   adaptable to placement of a removable reference electrode inside        or as close to the flow chamber as possible.    -   easy loading of beads.    -   manufacturable at acceptable cost    -   easy assembly of flow cell and attachment to the chip package.

Each of several example designs will be discussed, meeting thesecriteria. In each instance, one typically may choose to implement thedesign in one of two ways: either by attaching the flow cell to a frameand gluing the frame (or otherwise attaching it) to the chip or byintegrating the frame into the flow cell structure and attaching thisunified assembly to the chip. Further, designs may be categorized by theway the reference electrode is integrated into the arrangement.Depending on the design, the reference electrode may be integrated intothe flow cell (e.g., form part of the ceiling of the flow chamber) or bein the flow path (typically to the outlet or downstream side of the flowpath, after the sensor array).

A first example of a suitable experiment apparatus 3410 incorporatingsuch a fluidic interface is shown in FIGS. 34-37, the manufacture andconstruction of which will be discussed in greater detail below.

The apparatus comprises a semiconductor chip 3412 (indicated generally,though hidden) on or in which the arrays of wells and sensors areformed, and a fluidics assembly 3414 on top of the chip and deliveringthe sample to the chip for reading. The fluidics assembly includes aportion 3416 for introducing fluid containing the sample, a portion 3418for allowing the fluid to be piped out, and a flow chamber portion 3420for allowing the fluid to flow from inlet to outlet and along the wayinteract with the material in the wells. Those three portions areunified by an interface comprising a glass slide 3422 (e.g., ErieMicroarray Cat #C22-5128-M20 from Erie Scientific Company, Portsmouth,N.H., cut in thirds of size about 25 mm×25 mm).

Mounted on the top face of the glass slide are two fittings, 3424 and3426, such as nanoport fittings Part#N-333 from Upchurch Scientific ofOak Harbor, Wash. One port (e.g., 3424) serves as an inlet deliveringliquids from the pumping/valving system described below but not shownhere. The second port (e.g., 3426) is the outlet which pipes the liquidsto waste. Each port connects to a conduit 3428, 3432 such as flexibletubing of appropriate inner diameter. The nanoports are mounted suchthat the tubing can penetrate corresponding holes in the glass slide.The tube apertures should be flush with the bottom surface of the slide.

On the bottom of the glass slide, flow chamber 3420 may comprise variousstructures for promoting a substantially laminar flow across themicrowell array. For example, a series of microfluidic channels fanningout from the inlet pipe to the edge of the flow chamber may be patternedby contact lithography using positive photoresists such as SU-8photoresist from MicroChem. Corp. of Newton, Mass. Other structures willbe discussed below.

The chip 3412 will in turn be mounted to a carrier 3430, for packagingand connection to connector pins 3432.

For ease of description, to discuss fabrication starting with FIG. 38 weshall now consider the glass slide 3422 to be turned upside downrelative to the orientation it has in FIGS. 34-37.

A layer of photoresist 3810 is applied to the “top” of the slide (whichwill become the “bottom” side when the slide and its additional layersis turned over and mounted to the sensor assembly of ISFET array withmicrowell array on it). Layer 3810 may be about 150 μm thick in thisexample, and it will form the primary fluid carrying layer from the endof the tubing in the nanoports to the edge of the sensor array chip.Layer 3810 is patterned using a mask such as the mask 3910 of FIG. 39(“patterned” meaning that a radiation source is used to expose theresist through the mask and then the non-plasticized resist is removed).The mask 3910 has radiation-transparent regions which are shown as whiteand radiation-blocking regions 3920, which are shown in shading. Theradiation-blocking regions are at 3922-3928. The region 3926 will form achannel around the sensor assembly; it is formed about 0.5 mm inside theouter boundary of the mask 3920, to avoid the edge bead that is typical.The regions 3922 and 3924 will block radiation so that correspondingportions of the resist are removed to form voids shaped as shown. Eachof regions 3922, 3924 has a rounded end dimensioned to receive an end ofa corresponding one of the tubes 3428, 3432 passing through acorresponding nanoport 3424, 3426. From the rounded end, the regions3922, 3924 fan out in the direction of the sensor array to allow theliquid to spread so that the flow across the array will be substantiallylaminar. The region 3928 is simply an alignment pattern and may be anysuitable alignment pattern or be replaced by a suitable substitutealignment mechanism. Dashed lines on FIG. 38 have been provided toillustrate the formation of the voids 3822 and 3824 under mask regions3922 and 3924.

A second layer of photoresist is formed quite separately, not on theresist 3810 or slide 3422. Preferably it is formed on a flat, flexiblesurface (not shown), to create a peel-off, patterned plastic layer. Thissecond layer of photoresist may be formed using a mask such as mask4010, which will leave on the flexible substrate, after patterning, theborder under region 4012, two slits under regions 4014, 4016, whose usewill be discussed below, and alignment marks produced by patternedregions 4018 and 4022. The second layer of photoresist is then appliedto the first layer of photoresist using one alignment mark or set ofalignment marks, let's say produced by pattern 4018, for alignment ofthese layers. Then the second layer is peeled from its flexiblesubstrate and the latter is removed.

The other alignment mark or set of marks produced by pattern 4022 isused for alignment with a subsequent layer to be discussed.

The second layer is preferably about 150 μm deep and it will cover thefluid-carrying channel with the exception of a slit about 150 μm long ateach respective edge of the sensor array chip, under slit-formingregions 4014 and 4016.

Once the second layer of photoresist is disposed on the first layer, athird patterned layer of photoresist is formed over the second layer,using a mask such as mask 4110, shown in FIG. 41. The third layerprovides a baffle member under region 4112 which is as wide as thecollar 3310 on the sensor chip array (see FIG. 33) but about 300 μmnarrower to allow overlap with the fluid-carrying channel of the firstlayer. The third layer may be about 150 μm thick and will penetrate thechip collar 3310, toward the floor of the basin formed thereby, by 150μm. This configuration will leave a headspace of about 300 μm above thewells on the sensor array chip. The liquids are flowed across the wellsalong the entire width of the sensor array through the 150 μm slitsunder 4014, 4016.

FIG. 36 shows a partial sectional view, in perspective, of theabove-described example embodiment of a microfluidics and sensorassembly, also depicted in FIGS. 34 and 35, enlarged to make morevisible the fluid flow path. (A further enlarged schematic of half ofthe flow path is shown in FIG. 37.) Here, it will be seen that fluidenters via the inlet pipe 3428 in inlet port 3424. At the bottom of pipe3428, the fluid flows through the flow expansion chamber 3610 formed bymask area 3922, that the fluid flows over the collar 3310 and then downinto the bottom 3320 of the basin, and across the die 3412 with itsmicrowell array. After passing over the array, the fluid then takes avertical turn at the far wall of the collar 3310 and flows over the topof the collar to and across the flow concentration chamber 3612 formedby mask area 3924, exiting via outlet pipe 3432 in outlet port 3426.Part of this flow, from the middle of the array to the outlet, may beseen also in the enlarged diagrammatic illustration of FIG. 37, whereinthe arrows indicate the flow of the fluid.

The fluidics assembly may be secured to the sensor array chip assemblyby applying an adhesive to parts of mating surfaces of those twoassemblies, and pressing them together, in alignment.

Though not illustrated in FIGS. 34-36, the reference electrode may beunderstood to be a metallization 3710, as shown in FIG. 37, at theceiling of the flow chamber.

Another way to introduce the reference electrode is shown in FIG. 42.There, a hole 4210 is provided in the ceiling of the flow chamber and agrommet 4212 (e.g., of silicone) is fitted into that hole, providing acentral passage or bore through which a reference electrode 4220 may beinserted. Baffles or other microfeatures (not shown) may be patternedinto the flow channel to promote laminar flow over the microwell array.

FIGS. 43-44 show another alternative flow cell design, 4310. This designrelies on the molding of a single plastic piece or member 4320 to beattached to the chip to complete the flow cell. The connection to thefluidic system is made via threaded connections tapped into appropriateholes in the plastic piece at 4330 and 4340. Or, if the member 4320 ismade of a material such as polydimethylsiloxane (PDMS), the connectionsmay be made by simply inserting the tubing into an appropriately sizedhole is in the member 4320. A vertical cross section of this design isshown in FIGS. 43-44. This design may use an overhanging plastic collar4350 (which may be a solid wall as shown or a series of depending,spaced apart legs forming a downwardly extending fence-like wall) toenclose the chip package and align the plastic piece with the chippackage, or other suitable structure, and thereby to alignment the chipframe with the flow cell forming member 4320. Liquid is directed intothe flow cell via one of apertures 4330, 4340, thence downwardly towardsthe flow chamber.

In the illustrated embodiment, the reference electrode is introduced tothe top of the flow chamber via a bore 4325 in the member 4320. Theplacement of the removable reference electrode is facilitated by asilicone sleeve 4360 and an epoxy stop ring 4370 (see the blow-up ofFIG. 44). The silicone sleeve provides a tight seal and the epoxy stopring prevent the electrode from being inserted too far into the flowcell. Of course, other mechanisms may be employed for the same purposes,and it may not be necessary to employ structure to stop the electrode.And if a material such as PDMS is used for member 4320, the materialitself may form a watertight seal when the electrode is inserted,obviating need for the silicone sleeve.

FIGS. 45 and 46 show a similar arrangement except that member 4510 lacksa bore for receiving a reference electrode. Instead, the referenceelectrode 4515 is formed on or affixed to the bottom of central portion4520 and forms at least part of the flow chamber ceiling. For example, ametallization layer may be applied onto the bottom of central portion4520 before member 4510 is mounted onto the chip package.

FIGS. 47-48 show another example, which is a variant of the embodimentshown in FIGS. 43-44, but wherein the frame is manufactured as part ofthe flow cell rather attaching a flow port structure to a framepreviously attached to the chip surface. In designs of this type,assembly is somewhat more delicate since the wirebonds to the chip arenot protected by the epoxy encapsulating the chip. The success of thisdesign is dependent on the accurate placement and secure gluing of theintegrated “frame” to the surface of the chip. A counterpart embodimentto that of FIGS. 45-46, with the reference electrode 4910 on the ceilingof the flow chamber, and with the frame manufactured as part of the flowcell, is shown in FIGS. 49-50.

Yet another alternative for a fluidics assembly, as shown in FIGS.51-52, has a fluidics member 5110 raised by about 5.5 mm on stand-offs5120 from the top of the chip package 5130. This allows for an operatorto visually inspect the quality of the bonding between plastic piece5140 and chip surface and reinforce the bonding externally if necessary.

Some of the foregoing alternative embodiments also may be implemented ina hybrid plastic/PDMS configuration. For example, as shown in FIGS.53-54, a plastic part 5310 may make up the frame and flow chamber,resting on a PDMS “base” portion 5320. The plastic part 5310 may alsoprovides a region 5330 to the array, for expansion of the fluid flowfrom the inlet port; and the PDMS part may then include communicatingslits 5410, 5412 through which liquids are passed from the PDMS part toand from the flow chamber below.

The fluidic structure may also be made from glass as discussed above,such as photo-definable (PD) glass. Such a glass may have an enhancedetch rate in hydrofluoric acid once selectively exposed to UV light andfeatures may thereby be micromachined on the top-side and back-side,which when glued together can form a three-dimensional low aspect ratiofluidic cell.

An example is shown in FIG. 55. A first glass layer or sheet 5510 hasbeen patterned and etched to create nanoport fluidic holes 5522 and 5524on the top-side and fluid expansion channels 5526 and 5528 on theback-side. A second glass layer or sheet 5530 has been patterned andetched to provide downward fluid input/output channels 5532 and 5534, ofabout 300 μm height (the thickness of the layer). The bottom surface oflayer 5530 is thinned to the outside of channels 5532 and 5534, to allowthe layer 5530 to rest on the chip frame and protrusion area 5542 to beat an appropriate height to form the top of the flow channel. Two glasslayers, or wafers, and four lithography steps required. Both wafersshould be aligned and bonded (e.g., with an appropriate glue, not shown)such that the downward fluid input/output ports are aligned properlywith the fluid expansion channels. Alignment targets may be etched intothe glass to facilitate the alignment process.

Nanoports may be secured over the nanoport fluidic holes to facilitateconnection of input and output tubing.

A central bore 5550 may be etched through the glass layers for receivinga reference electrode, 5560. The electrode may be secured and sealed inplace with a silicone collar 5570 or like structure; or the electrodemay be equipped integrally with a suitable washer for effecting the samepurpose.

By using glass materials for the two-layer fluidic cell, the referenceelectrode may also be a conductive layer or pattern deposited on thebottom surface of the second glass layer (not shown). Or, as shown inFIG. 56, the protrusion region may be etched to form a permeable glassmembrane 5610 on the top of which is coated a silver (or other material)thin-film 5620 to form an integrated reference electrode. A hole 5630may be etched into the upper layer for accessing the electrode and ifthat hole is large enough, it can also serve as a reservoir for a silverchloride solution. Electrical connection to the thin-film silverelectrode may be made in any suitable way, such as by using a clip-onpushpin connector or alternatively wirebonded to the ceramic ISFETpackage.

Still another example embodiment for a fluidic assembly is shown inFIGS. 57-58. This design is limited to a plastic piece 5710 whichincorporates the frame and is attached directly to the chip surface, andto a second piece 5720 which is used to connect tubing from the fluidicsystem and similarly to the PDMS piece discussed above, distributes theliquids from the small bore tube to a wide flat slit. The two pieces areglued together and multiple (e.g., three) alignment markers (not shown)may be used to precisely align the two pieces during the gluing process.A hole may be provided in the bottom plate and the hole used to fill thecavity with an epoxy (for example) to protect the wirebonds to the chipand to fill in any potential gaps in the frame/chip contact. In theillustrated example, the reference electrode is external to the flowcell (downstream in the exhaust stream, through the outlet port—seebelow), though other configurations of reference electrode may, ofcourse, be used.

Still further examples of flow cell structures are shown in FIGS. 59-60.FIG. 59A comprises eight views (A-H) of an injection molded bottomlayer, or plate, 5910, for a flow cell fluidics interface, while FIG.59B comprises seven views (A-G) of a mating, injection molded top plate,or layer, 5950. The bottom of plate 5910 has a downwardly depending rim5912 configured and arranged to enclose the sensor chip and an upwardlyextending rim 5914 for mating with the top plate 5610 along its outeredge. To form two fluid chambers (an inlet chamber and an outletchamber) between them. A stepped, downwardly depending portion 5960 oftop plate 5950, separates the input chamber from the output chamber. Aninlet tube 5970 and an outlet tube 5980 are integrally molded with therest of top plate 5950. From inlet tube 5970, which empties at the smallend of the inlet chamber formed by a depression 5920 in the top of plate5910, to the outlet edge of inlet chamber fans out to direct fluidacross the whole array.

Whether glass or plastic or other material is used to form the flowcell, it may be desirable, especially with larger arrays, to include inthe inlet chamber of the flow cell, between the inlet conduit and thefront edge of the array, not just a gradually expanding (fanning out)space, but also some structure to facilitate the flow across the arraybeing suitably laminar. Using the bottom layer 5990 of an injectionmolded flow cell as an example, one example type of structure for thispurpose, shown in FIG. 59C, is a tree structure 5992 of channels fromthe inlet location of the flow cell to the front edge of the microwellarray or sensor array, which should be understood to be under the outletside of the structure, at 5994.

There are various other ways of providing a fluidics assembly fordelivering an appropriate fluid flow across the microwell and sensorarray assembly, and the forgoing examples are thus not intended to beexhaustive.

Reference Electrode

Commercial flow-type fluidic electrodes, such as silver chlorideproton-permeable electrodes, may be inserted in series in a fluidic lineand are generally designed to provide a stable electrical potentialalong the fluidic line for various electrochemical purposes. In theabove-discussed system, however, such a potential must be maintained atthe fluidic volume in contact with the microwell ISFET chip. Withconventional silver chloride electrodes, it has been found difficult,due to an electrically long fluidic path between the chip surface andthe electrode (through small channels in the flow cell), to achieve astable potential. This led to reception of noise in the chip'selectronics. Additionally, the large volume within the flow cavity ofthe electrode tended to trap and accumulate gas bubbles that degradedthe electrical connection to the fluid. With reference to FIG. 60, asolution to this problem has been found in the use of a stainless steelcapillary tube electrode 6010, directly connected to the chip's flowcell outlet port 6020 and connected to a voltage source (not shown)through a shielded cable 6030. The metal capillary tube 6010 has a smallinner diameter (e.g., on the order of 0.01″) that does not trap gas toany appreciable degree and effectively transports fluid and gas likeother microfluidic tubing. Also, because the capillary tube can bedirectly inserted into the flow cell port 6020, it close to the chipsurface, reducing possible electrical losses through the fluid. Thelarge inner surface area of the capillary tube (typically about 2″ long)may also contribute to its high performance. The stainless steelconstruction is highly chemically resistant, and should not be subjectto electrochemical effects due to the very low electrical current use inthe system (<1 μA). A fluidic fitting 6040 is attached to the end of thecapillary that is not in the flow cell port, for connection to tubing tothe fluid delivery and removal subsystem.

Fluidics System

A complete system for using the sensor array will include suitable fluidsources, valving and a controller for operating the valving to lowreagents and washes over the microarray or sensor array, depending onthe application. These elements are readily assembled from off-the-shelfcomponents, with and the controller may readily be programmed to performa desired experiment.

As already discussed, the apparatus and systems of the invention can beused to detect and/or monitor interactions between various entities.These interactions include chemical or enzymatic reactions in whichsubstrates and/or reagents are consumed and/or reaction byproducts aregenerated. An example of an enzymatic reaction that can be monitoredaccording to the invention is nucleic acid sequencing, which will bediscussed in greater detail herein. In the context of a sequencingreaction, the apparatus and system provided herein is able to detectnucleotide incorporation based on changes in the chemFET current.Current changes may be the result of one or more of the following eventseither singly or some combination thereof: generation of PPi, generationof Pi (e.g., in the presence of pyrophosphatase), generation of hydrogen(and concomitant changes in pH for example in the presence of lowstrength buffer), reduced concentration of unincorporated dNTP at thechemFET surface, delayed arrival of dNTP at the chemFET surface, and thelike. It is to be understood that the methods provided herein are notdependent upon the mechanism by which the current change is effected.And accordingly, the invention contemplates sequencing of nucleic acidsbased on changes in the chemFET current. The methods provided herein inregards to sequencing can be contrasted to those in the literatureincluding Pourmand et al. PNAS 2006 103(17):6466-6470.

FIG. 61 illustrates the production of PPi resulting from theincorporation of a nucleotide in a newly synthesized nucleic acidstrand. PPi generated as a reaction byproduct of nucleotideincorporation in a nucleic acid strand can be detected directly even inthe absence of a PPi receptor (such as those provided in FIG. 11B) andin the absence of a detectable pH change (e.g., as may occur in thepresence of a strong buffer, as defined herein). The simple presence ofPPi is sufficient, in some instances, to cause an electrical change inthe chemFET surface, thereby resulting in a current change. The currentchange may result from PPi generation alone or in combination with otherevents such as those described above.

Thus, in one aspect, the invention contemplates sequencing nucleic acidsusing a chemFET array such as an ISFET array. The method of theinvention is a “sequencing by synthesis” method since it requiressynthesis of a new nucleic acid strand that is complementary to thestrand being sequenced.

The release of PPi following incorporation of a nucleotide in a newlysynthesized nucleic acid strand is shown in FIG. 61. The incorporationof a dNTP into the nucleic acid strand releases PPi which can then behydrolyzed to two orthophosphates (Pi) and one hydrogen ion. Thegeneration of the hydrogen ion therefore can facilitate detection ofnucleotide incorporation on the basis of pH change. Alternatively, asdiscussed herein, PPi generation (as detected in the absence or presenceof PPi receptors) can facilitate detection of nucleotide incorporationon the basis of pH change. And in still another embodiment, PPi may beconverted to Pi using pyrophosphatase and Pi may be detected directly orindirectly. Any and all of these events (and more as described herein)may be involved in causing a current change in the chemFET thatcorrelates with nucleotide incorporation.

The sequencing reactions aim to maximize complete incorporation acrossall wells for any given dNTP, reduce or decrease the number ofunincorporated dNTPs that remain in the wells, and achieve as a high asignal to noise ratio as possible.

The nucleic acid being sequenced is referred to herein as the targetnucleic acid. Target nucleic acids include but are not limited to DNAsuch as but not limited to genomic DNA, mitochondrial DNA, cDNA and thelike, and RNA such as but not limited to mRNA, miRNA, and the like. Thenucleic acid may be from any source including naturally occurringsources or synthetic sources. The nucleic acids may be PCR products,cosmids, plasmids, naturally occurring or synthetic libraries, and thelike. The invention is not intended to be limited in this regard. Themethods provided herein can be used to sequence nucleic acids of anylength. To be clear, the Examples provide a proof of principledemonstration of the sequencing of four templates of known sequence.This artificial model is intended to show that the apparatus and systemare able to readout nucleotide incorporation that correlates to theknown sequence of the templates. This is not intended to representtypical use of the method or system in the field. The following is abrief description of these methods.

Target nucleic acids are prepared using any manner known in the art. Asan example, genomic DNA may be harvested from a sample according totechniques known in the art (see for example Sambrook'et al.“Maniatis”). Following harvest, the DNA may be fragmented to yieldnucleic acids of smaller length. The resulting fragments may be on theorder of hundreds, thousands, or tens of thousands nucleotides inlength. In some embodiments, the fragments are 200-1000 base pairs insize, or 300-800 base pairs in size, although they are not so limited.Nucleic acids may be fragmented by any means including but not limitedto mechanical, enzymatic or chemical means. Examples include shearing,sonication, nebulization and endonuclease (e.g., Dnase I) digestion, orany other technique known in the art to produce nucleic acid fragments,preferably of a desired length. Fragmentation can be followed by sizeselection techniques can be used enrich or isolate fragments of aparticular length or size. Such techniques are also known in the art andinclude but are not limited to gel electrophoresis or SPRI.

In some embodiments, the size selected target nucleic acids are ligatedto adaptor sequences on both the 5′ and 3′ ends. These adaptor sequencescomprise amplification primer sequences to be used in amplifying thetarget nucleic acids. One adaptor sequence may also comprise a sequencecomplementary to the sequencing primer. The opposite adaptor sequencemay comprise a moiety that facilitates binding of the nucleic acid to asolid support such as but not limited to a bead. An example of such amoiety is a biotin molecule (or a double biotin moiety, as described byDiehl et al. Nature Methods, 2006, 3(7):551-559) and such a labelednucleic acid can therefore be bound to a solid support having avidin orstreptavidin groups. The resulting nucleic acid is referred to herein asa template nucleic acid. The template nucleic acid comprises at leastthe target nucleic acid and usually comprises nucleotide sequences inaddition to the target.

In some instances a spacer is used to distance the template nucleic acid(and in particular the target nucleic acid sequence comprised therein)from the bead. This facilitates sequencing of the end of the targetclosest to the bead. Examples of suitable linkers are known in the art(see Diehl et al. Nature Methods, 2006, 3(7):551-559) and include butare not limited to carbon-carbon linkers such as but not limited toiSp18.

The solid support to which the template nucleic acids are bound isreferred to herein as the “capture solid support”. If the solid supportis a bead, then such bead is referred to herein as a “capture bead”. Thebeads can be made of any material including but not limited tocellulose, cellulose derivatives, gelatin, acrylic resins, glass, silicagels, polyvinyl pyrrolidine (PVP), co-polymers of vinyl and acrylamide,polystyrene, polystyrene cross-linked with divinylbenzene or the like(see, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamides,latex gels, dextran, crosslinked dextrans (e.g., Sephadex™), rubber,silicon, plastics, nitrocellulose, natural sponges, metal, and agarosegel (Sepharosem™). In one embodiment, the beads are streptavidin-coatedbeads. The bead diameter will depend on the density of the ISFET andwell array used with larger arrays (and thus smaller sized wells)requiring smaller beads. Generally the bead size may be about 1-10 μM,and more preferably 2-6 μM. In some embodiments, the beads are about5.91 μM while in other embodiments the beads are about 2.8 μM. It is tobe understood that the beads may or may not be perfectly spherical inshape. It is to be understood that other beads may be used and othermechanisms for attaching the nucleic acid to the beads may be utilized.

As discussed herein, the sequencing reactions are carried out in wellsthat are situated above the chemFETs. The wells (referred to hereininterchangeably as reaction chambers or microwells) may vary in sizebetween arrays. Preferably the width to height ratio of the well is 1:1to 1:1.5. The bead to well size is preferably in the range of 0.6-0.8.

A homogeneous population of amplified nucleic acids are conjugated toone or more beads with the proviso that each bead will ultimately bebound to a plurality of identical nucleic acid sequences. The degree ofloading of nucleic acid templates onto beads will depend on a number offactors including the bead size and the length of the nucleic acid. Inmost aspects, maximal loading of the beads is desired. Amplification andconjugation of nucleic acids to solid support such as beads may beaccomplished in a number of ways, including but not limited to emulsionPCR as described by Margulies et al. Nature 2005 437(15):376-380 andaccompanying supplemental materials. In some embodiments, theamplification is a representative amplification. A representativeamplification is an amplification that does not alter the relativerepresentation of any nucleic acid species.

Before and/or while in the wells of the flow chamber, the beads areincubated with a sequencing primer that binds to its complementarysequence located on the 3′ end of the template nucleic acid (i.e.,either in the amplification primer sequence or in another adaptorsequence ligated to the 3′ end of the target nucleic acid) and with apolymerase for a time and under conditions that promote hybridization ofthe primer to its complementary sequence and that promote binding of thepolymerase to the template nucleic acid. The primer can be of virtuallyany sequence provided it is long enough to be unique. The hybridizationconditions are such that the primer will hybridize to only its truecomplement on the 3′ end of the template. Suitable conditions aredisclosed in Margulies et al. Nature 2005 437(15):376-380 andaccompanying supplemental materials.

Suitable polymerases include but are not limited to DNA polymerase, RNApolymerase, or a subunit thereof, provided it is capable of synthesizinga new nucleic acid strand based on the template and starting from thehybridized primer. An example of a suitable polymerase subunit is theexo-version of the Klenow fragment of E. coli DNA polymerase I whichlacks 3′ to 5′ exonuclease activity. The enzyme is therefore bound tothe bead (or corresponding solid support) but not to the ISFET surfaceitself. The template nucleic acid is also contacted with other reagentsand/or cofactors including but not limited to buffer, detergent,reducing agents such as dithiothrietol (DTT, Cleland's reagent), singlestranded binding proteins, and the like before and/or while in the well.En one embodiment, the template nucleic acid is contacted with theprimer and the polymerase prior to its introduction into the flowchamber and wells thereof.

The nucleic acid loaded beads are introduced into the flow chamber andultimately the wells situated above the ISFET array. The method requiresthat each well in the flow chamber contain only one nucleic acid loadedbead since the presence of two beads per well will yield one unusablesequencing information derived from two different nucleic acids. TheExamples provides a brief description of an exemplary bead loadingprotocol in the context of magnetic beads. It is to be understood that asimilar approach could be used to load other bead types. The protocolhas been demonstrated to reduce the likelihood and incidence of trappedair in the wells of the flow chamber; uniformly distribute nucleic acidloaded beads in the totality of wells of the flow chamber, and avoid thepresence and/or accumulation of excess beads in the flow chamber.

The percentage of occupied wells on the chip may vary depending on themethods being performed. If the method is aimed at extracting maximumsequence data in the shortest time possible, then higher occupancy isdesirable. If speed and throughout is not as critical, then loweroccupancy may be tolerated. Therefore depending on the embodiment,suitable occupancy percentages may be at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or 100% of the wells. As used herein, occupancyrefers to the presence of one nucleic acid loaded bead in a well and thepercentage occupancy refers to the proportion of total wells on a chipthat are occupied by a single bead. Wells that are occupied by more thanone bead cannot be used in the analyses contemplated by the invention.

Ultimately a homogeneous population of template nucleic acids is placedinto one or more of a plurality of wells, each well situated over andthus corresponding to at least one ISFET. As discussed above, preferablythe well contains at least 10, at least 100, at least 1000, at least10⁴, at least 10⁵, at least 10⁶, or more copies of an identical templatenucleic acid. Identical template nucleic acids means at a minimum thatthe templates are identical in sequence. Most and preferably all thetemplate nucleic acids within a well are uniformly hybridized to aprimer. Uniform hybridization of the template nucleic acids to theprimers means that the primer hybridizes to the template at the samelocation (i.e., the sequence along the template that is complementary tothe primer) as every other primer-template hybrid in the well. Theuniform positioning of the primer on every template allows theco-ordinated synthesis of all new nucleic acid strands within a well,thereby resulting in a greater signal-to-noise ratio.

Nucleotides are then added in flow, or by any other suitable method, insequential order to the flow chamber and thus the wells. The nucleotidescan be added in any order provided it is known and for the sake ofsimplicity kept constant throughout a run. If the incorporation ofnucleotides is based on detection of PPi rather than detection of pHchange as a result of PPi release, then it is preferable to maintain arelatively constant level and concentration of nucleotides throughoutthe reactions and washes. One way of accomplishing this is to add ATP tothe wash buffer such that dNTPs flowing into a well are displacing ATPfrom the well. The ATP matches the ionic strength of the dNTPs enteringthe wells and it also has a similar diffusion profile as those dNTPs. Inthis way, influx and efflux of dNTPs during the sequencing reaction donot interfere with measurements at the chemFET. The concentration of ATPused is on the order of the concentration of dNTP used.

A typical sequencing cycle proceeds as follows: washing of the flowchamber (and wells) with wash buffer containing ATP, introduction of afirst dNTP species (e.g., dATP) into the flow chamber (and wells),release and detection of PPi (by any of the mechanisms describedherein), washing of the flow chamber (and wells) with wash buffercontaining ATP, washing of the flow chamber (and wells) with wash buffercontaining apyrase, washing of the flow chamber (and wells) with washbuffer containing ATP, and introduction of a second dNTP species. Thisprocess is continued until all 4 dNTP (i.e., dATP, dCTP, dGTP and dTTP)have been flowed through the chamber and allowed to incorporate into thenewly synthesized strands. This 4-nucleotide cycle may be repeated anynumber of times including but not limited to 10, 25, 50, 100, 200 ormore times. The number of cycles will be governed by the length of thetemplate being sequenced and the need to replenish reaction reagents, inparticular the dNTP stocks and wash buffers.

As part of the sequencing reaction, a dNTP will be ligated to (or“incorporated into” as used herein) the 3′ of the newly synthesizedstrand (or the 3′ end of the sequencing primer in the case of the firstincorporated dNTP) if its complementary nucleotide is present at thatsame location on the template nucleic acid. Incorporation of theintroduced dNTP (and concomitant release of PPi) therefore indicates theidentity of the corresponding nucleotide in the template nucleic acid.If no change in the electric field is detected by the ISFET, then thedNTP has not been incorporated and one can conclude that thecomplementary nucleotide was not present in the template at thatlocation. If a change in the electric field is detected, then theintroduced dNTP has been incorporated into the newly synthesized strand.There is a positive correlation between dNTP incorporation and PPirelease and the response of the ISFET, and as a result it is furtherpossible to quantitate the number of dNTP incorporated. In other words,the voltage change registered at the ISFET is related to the number ofdNTP incorporated. The result is that no sequence information is lostthrough the sequencing of a homopolymer stretch (e.g., poly A, poly T,poly C, or poly G) in the template. As an example, if the templatenucleic acid includes a sequence of 5′ CAAAAG 3′, the ISFET willregister a signal (e.g., in terms of millivolt change) upon introductionof dCTP, and then it will register a signal of greater magnitude uponthe introduction of dTTP, followed by another signal upon theintroduction of dGTP. The magnitude of the signals arising uponintroduction of dCTP and dTTP will be essentially equivalent and willcorrelate with the millivoltage change resulting from a singlenucleotide incorporation. The magnitude of the signal arising uponintroduction of dTTP will be greater than the signals arising fromsingle dNTP incorporation. The magnitude of these signals may beadditive, and depending on the length of the homopolymer stretch may notbe readily apparent in a voltage versus time (or frame) plot (such asthose shown in FIG. 71A-D, right panels). Signals may be measured usingpeak intensity or area under the curve from a voltage versus time (orframe) plot.

Apyrase is an enzyme that degrades residual unincorporated nucleotidesconverting them into monophosphate and releasing inorganic phosphate inthe process. It is useful for degrading dNTPs that are not incorporatedand/or that are in excess in any and all wells. It is important thatexcess and/or unreacted dNTP be washed away from any and all wellsbefore introduction of the subsequent dNTP. Accordingly, addition ofapyrase during the synthesis reaction and between the introduction ofdifferent dNTPs is useful to remove excess dNTPs that would otherwiseobscure the sequencing data.

Additional sequencing reaction reagents such as those described abovemay be introduced throughout the reaction, although in some cases thismay not be necessary. For example additional polymerase, DTT, SBB andthe like may be added if necessary.

The invention therefore contemplates performing a plurality of differentsequencing reactions simultaneously. A plurality of identical sequencingreactions is occurring in each occupied well simultaneously. It is thissimultaneous and identical incorporation of dNTP within each well thatincreases the signal to noise ratio, thereby making detection of thesequencing reaction byproduct possible. By performing sequencingreactions in a plurality of wells simultaneously, a plurality ofdifferent sequencing reactions are also performed simultaneously.

The sequencing reaction can be run at a range of temperatures.Typically, the reaction is run in the range of 30-60° C., 35-55° C., or40-45° C. It is preferable to run the reaction at temperatures thatprevent formation of secondary structure in the nucleic acid. Howeverthis must be balanced with the binding of the primer (and the newlysynthesized strand) to the template nucleic acid and the reducedhalf-life of apyrase at higher temperatures. A suitable temperature isabout 41° C. The solutions including the wash buffers and the dNTPsolutions are generally warmed to these temperatures in order not toalter the temperature in the wells. The wash buffer containing apyrasehowever is preferably maintained at a lower temperature in order toextend the half-life of the enzyme. Typically, this solution ismaintained at about 4-15° C., and more preferably 4-10° C.

The nucleotide incorporation reaction can occur very rapidly. As aresult, it may be desirable in some instances to slow the reaction downin order to ensure maximal data capture during the reaction. Thediffusion of reagents and/or byproducts can be slowed down in a numberof ways including but not limited to addition of packing beads in thewells. The packing beads also tend to increase the concentration ofreagents and/or byproducts at the chemFET surface, thereby increasingthe potential for signal. The presence of packing beads generally allowsa greater time to sample (e.g., by 2- or 4-fold).

Data capture rates can vary and be for example anywhere from 10-100frames per second and the choice of which rate to use will be dictatedat least in part by the well size and the presence of packing beads.Smaller well sizes generally require faster data capture rates.

In some aspects of the invention that are flow-based and where the topface of the well is open and in communication with fluid over theentirety of the chip, it is important to detect the released PPi orother byproduct (e.g., H⁺) prior to diffusion out of the well. Diffusionof either reaction byproduct out of the well will lead to falsenegatives (because the byproduct is not detected in that well) andpotential false positives in adjacent or downstream wells, and thusshould be avoided. Packing beads may also help reduce the degree ofdiffusion and/or cross-talk between wells.

Thus in some embodiments packing beads are used in addition to thenucleic acid-loaded beads. The packing beads may be magnetic (includingsuperparamagnetic) but they are not so limited. In some embodiments thepacking beads and the capture beads are made of the same material (e.g.,both are magnetic, both are polystyrene, etc.), while in otherembodiment they are made of different materials (e.g., the packing beadsare polystyrene and the capture beads are magnetic). The packing beadsare generally smaller than the capture beads. The difference in size maybe vary and may be 5-fold, 10-fold, 15-fold, 20-fold or more. As anexample, 0.35 μm diameter packing beads have been used with 5.91 μmcapture beads. Such packing beads are commercially available fromsources such as Bang Labs. The placement of the packing beads relativeto the capture bead may vary. For example, the packing beads maysurround the capture bead and thereby prevent the capture bead fromcontacting the ISFET surface. As another example, the packing beads maybe loaded into the wells following the capture beads in which case thecapture bead is in contact with the ISFET surface. The presence ofpacking beads between the capture bead and the ISFET surface slows thediffusion of the sequencing byproducts such as PPi, thereby facilitatingdata capture.

The invention further contemplates the use of packing beads ormodifications to the chemFET surface (as described herein) to preventcontact and thus interference of the chemFET surface with the templatenucleic acids bound to the capture beads. A layer of packing beads thatis 0.1-0.5 μm in depth or height would preclude this interaction.

The sequencing reaction may be preceded by an analysis of the arrays todetermine the location of beads. It has been found that in the absenceof flow the background signal (i.e., noise) is less than or equal toabout 0.25 mV, but that in the presence of DNA-loaded capture beads thatsignal increases to about 1.0 mV+/−0.5 mV. This increase is sufficientto allow one to determine wells with beads.

The invention further contemplates kits comprising the various reagentsnecessary to perform a sequencing reaction and instructions of useaccording to the methods set forth herein.

One preferred kit comprises one or more containers housing wash buffer,one or more containers each containing one of the following reagents:dATP buffer, dCTP buffer, dGTP buffer or dTTP buffer, dATP, dCTP, dGTPand dTTP stocks, apyrase, SSB, polymerase, packing beads and optionallypyrophosphatase. Importantly the kits comprise only naturally occurringdNTPs.

It is to be understood that interactions between receptors and ligandsor between two members of a binding pair or between components of amolecular complex can also be detected using the chemFET arrays.Examples of such interactions include hybridization of nucleic acids toeach other, protein-nucleic acid binding, protein-protein binding,enzyme substrate binding, enzyme-inhibitor binding, antigen-antibodybinding, and the like. Any binding or hybridization event that causes achange of the semiconductor charge density at the FET interface and thuschanges the current that flows from the source to the drain of thesensors described herein can be detected according to the invention.

In these embodiments, the passivation layer (or possibly an intermediatelayer coated onto the passivation layer) is functionalized with nucleicacids (e.g., DNA, RNA, miRNA, cDNA, and the like), antigens (which canbe of any nature), proteins (e.g., enzymes, cofactors, antibodies,antibody fragments, and the like), and the like. Conjugation of theseentities to the passivation layer can be direct or indirect (e.g., usingbifunctional linkers that bind to both the passivation layer reactivegroup and the entity to be bound).

As an example, reaction groups such as amine or thiol groups may beadded to a nucleic acid at any nucleotide during synthesis to provide apoint of attachment for a bifunctional linker. As another example, thenucleic acid may be synthesized by incorporating conjugation-competentreagents such as Uni-Link AminoModifier, 3′-DMT-C6-Amine-ON CPG,AminoModifier II, N-TFA-C6-AminoModifier, C6-ThiolModifier, C6-DisulfidePhosphoramidite and C6-Disulfide CPG (Clontech, Palo Alto, Calif.).Other methods for attaching nucleic acids are discussed below.

In one aspect of the invention, the chemFET arrays are provided incombination with nucleic acid arrays. Nucleic acids in the form of shortnucleic acids (e.g., oligonucleotides) or longer nucleic acids (e.g.,full length cDNAs) can be provided on chemFET surfaces of the arraysdescribed herein. Nucleic acid arrays generally comprise a plurality ofphysically defined regions on a planar surface (e.g., “spots”) each ofwhich has conjugated to it one and more preferably more nucleic acids.The nucleic acids are usually single stranded. The nucleic acidsconjugated to a given spot are usually identical. In the context of anoligonucleotide array, these nucleic acids may be on the order of less100 nucleotides in length (including about 10, 20, 25, 30, 40, 50, 60,70, 80, 90 or 100 nucleotides in length). If the arrays are used todetect certain genes (including mutations in such genes or expressionlevels of such genes), then the array may include a number of spots eachof which contains oligonucleotides that span a defined and potentiallydifferent sequence of the gene. These spots are then located across theplanar surface in order to exclude position related effects in thehybridization and readout means of the array.

The arrays are contacted with a sample being tested. The sample may be agenomic DNA sample, a cDNA sample from a cell, a tissue or a mass (e.g.,a tumor), a population of cells that are grown on the array, potentiallyin a two dimensional array that corresponds to the underlying sensorarray, and the like. Such arrays are therefore useful for determiningpresence and/or level of a particular gene or of its expression,detecting mutations within particular genes (such as but not limited todeletions, additions, substitutions, including single nucleotidepolymorphisms), and the like.

The binding or hybridization of the sample nucleic acids and theimmobilized nucleic acids is generally performed under stringenthybridization conditions as that term is understood in the art. (See forexample Sambrook et al. “Maniatis”.) Examples of relevant conditionsinclude (in order of increasing stringency): incubation temperatures of25° C., 37° C., 50° C. and 68° C.; buffer concentrations of 10×SSC,6×SSC, 4×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citratebuffer) and their equivalents using other buffer systems; formamideconcentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutesto 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2,or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionizedwater. By way of example hybridization may be performed at 50% formamideand 4×SSC followed by washes of 2×SSC/formamide at 50° C. and with1×SSC.

Nucleic acid arrays include those in which already formed nucleic acidssuch as cDNAs are deposited (or “spotted”) on the array in a specificlocation. Nucleic acids can be spotted onto a surface bypiezoelectrically deposition, UV crosslinking of nucleic acids topolymer layers such as but not limited to poly-L-lysine or polypyrrole,direct conjugation to silicon coated SiO₂ as described in published USpatent application 2003/0186262, direct conjugation to a silanisedchemFET surface (e.g., a surface treated with3-aminopropyltriethoxysilane (APTES) as described by Uslu et al.Biosensors and Bioelectronics 2004, 19: 1723-1731, for example.

Nucleic acid arrays also include those in which nucleic acids (such asoligonucleotides of known sequence) are synthesized directly on thearray. Nucleic acids can be synthesized on arrays using art-recognizedtechniques such as but not limited to printing with fine-pointed pinsonto glass slides, photolithography using pre-made masks,photolithography using dynamic micromirror devices (such as DLPmirrors), ink-jet printing, or electrochemistry on microelectrodearrays. Reference can also be made to Nuwaysir et al. 2002 “Geneexpression analysis using oligonucleotide arrays produced by masklessphotolithography.”. Genome Res 12: 1749-1755. Commercial sources of thislatter type of array include Agilent, Affymetrix, and NimbleGen.

Thus the chemFET passivation layer may be coated with an intermediatelayer of reactive molecules (and therefore reactive groups) to which thenucleic acids are bound and/or from which they are synthesized.

The invention contemplates combining such nucleic acid arrays with thechemFET arrays and particularly the “large scale” chemFET arraysdescribed herein. The chemFET/nucleic acid array can be used in avariety of applications, some of which will not require the wells (ormicrowells or reaction chambers, as they are interchangeably referred toherein). Since analyses may still be carried out in flow, including in a“closed” system (i.e., where the flow of reagents and wash solutions andthe like is automated), there will be one or more flow chambers situatedabove and in contact with the array. The use of multiple flow chambersallows multiple samples (or nucleic acid libraries) to be analyzedsimultaneously. There may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more flowchambers. This configuration applies equally to other biological arraysincluding those discussed herein such as protein arrays, antibodyarrays, enzyme arrays, chemical arrays, and the like.

Since the binding event between binding partners or between componentsof a complex is detected electronically via the underlying FET, suchassays may be carried out without the need to manipulate (e.g.,extrinsically label) the sample being assayed. This is advantageoussince such manipulation invariably results in loss of sample andgenerally requires increased time and work up. In addition, the presentmethod allows binding interactions to be studied in real time.

Protein arrays used in combination with the chemFET arrays of theinvention are also contemplated. Protein arrays comprise proteins orpeptides or other amino acid comprising biological moiety bound to aplanar surface in an organized and predetermined manner. Such proteinsinclude but are not limited to enzymes, antibodies and antibodyfragments or antibody mimics (e.g., single chain antibodies).

In one embodiment, a protein array may comprise a plurality of differentproteins (or other amino acid containing biological moieties). Eachprotein, and preferably a plurality of proteins, is present in apredetermined region or “cell” of the array. The regions (or cells) arealigned with the sensors in the sensor array such that there is onesensor for each region (or cell). The plurality of proteins in a singleregion (or cell) may vary depending on the size of the protein and thesize of the region (or cell) and may be but is not limited to at least10, 50, 100, 500, 10³, 10⁴ or more. The array itself may have any numberof cells, including but not limited to at least 10, 10², 10³, 10⁴, 10⁵,10⁶, 10⁷, or more. In one application, the array is exposed to a samplethat is known to contain or is suspected of containing an analyte thatbinds to the protein. The analyte may be a substrate or an inhibitor ifthe protein is an enzyme. The analyte may be any molecule that binds tothe protein including another protein, a nucleic acid, a chemicalspecies (whether synthetic or naturally occurring), and the like.

It is to be understood that, like the nucleic acid arrays contemplatedherein, the readout from the protein arrays will be a change in currentthrough the chemFET and thus no additional step of labeling and/or labeldetection is required in these array methods.

In another embodiment, the protein array may comprise a plurality ofidentical proteins (or other amino acid containing biological moieties).The identical proteins may be uniformly distributed on a planar surfaceor they may be organized into discrete regions (or cells) on thatsurface. In these latter embodiments, the regions (or cells) are alignedwith the sensors in the sensor array such that there is one sensor foreach region (or cell).

The proteins may be synthesised off-chip, then purified and attached tothe array. Alternatively they can be synthesised on-chip, similarly tothe nucleic acids discussed above. Synthesis of proteins using cell-freeDNA expression or chemical synthesis is amenable to on-chip synthesis.Using cell-free DNA expression, proteins are attached to the solidsupport once synthesized. Alternatively, proteins may be chemicallysynthesized on the solid support using solid phase peptide synthesis.Selective deprotection is carried out through lithographic methods or bySPOT-synthesis. Reference can be made to at least MacBeath andSchreiber, Science, 2000, 289:1760-1763, or Jones et al. Nature, 2006,439:168-174. Reference can also be made to U.S. Pat. No. 6,919,211 toFodor et al.

Chemical compound microarrays in combination with chemFET arrays arealso envisioned. Chemical compound microarrays can be made by covalentlyimmobilizing the compounds (e.g., organic compounds) on the solidsurface with diverse linking techniques (may be referred to in theliterature as “small molecule microarray”), by spotting and dryingcompounds (e.g., organic compounds) on the solid surface withoutimmobilization (may be referred to in the literature as “micro arrayedcompound screening (μARCS)”), or by spotting organic compounds in ahomogenous solution without immobilization and drying effect(commercialized as DiscoveryDot™ technology by Reaction BiologyCorporation).

Tissue microarrays in combination with chemFET arrays are furthercontemplated by the invention. Tissue microarrays are discussed ingreater detail in Battifora Lab Invest 1986, 55:244-248; Battifora andMehta Lab Invest 1990, 63:722-724; and Kononen et al. Nat Med 1998,4:844-847.

The configurations of the chemFET arrays and the biological or chemicalarrays are similar in each instance and the discussion of onecombination array will apply to others described herein or otherwiseknown in the art.

In yet another aspect, the invention contemplates analysis of cellcultures (e.g., two-dimensional cells cultures) (see for example Baumannet al. Sensors and Actuators B 55 1999 77:89), and tissue sectionsplaced in contact with the chemFET array. As an example, a brain sectionmay be placed in contact with the chemFET array of the invention andchanges in the section may be detected either in the presence or absenceof stimulation such as but not limited to neurotoxins and the like.Transduction of neural processes and/or stimulation can thereby beanalyzed. In these embodiments, the chemFETs may operate by detectingcalcium and/or potassium fluxes via the passivation layer itself or viareceptors for these ions that are coated onto the passivation layer.

In yet another aspect, the invention contemplates the use of chemFETarrays, functionalized as described herein or in another manner, for usein vivo. Such an array may be introduced into a subject (e.g., in thebrain or other region that is subject to ion flux) and then analyzed forchanges based on the status of the subject.

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than'A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

EXAMPLES

The following is an example of a proof of principle for rapid sequencingof single-stranded oligonucleotides using an ISFET array.

1.1. Binding of Single-Stranded Oligonucleotides to Streptavidin-CoatedMagnetic Beads.

Single-stranded DNA oligonucleotide templates with a 5′ Dual Biotin tag(HPLC purified), and a 20-base universal primer were ordered from IDT(Integrated DNA Technologies, Coralville, Ind.). Templates were 60 basesin length, and were designed to include 20 bases at the 3′ end that werecomplementary to the 20-base primer (Table 1, italics). The lyophilizedand biotinylated templates and primer were re-suspended in TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8) as 40 μM stock solutions and as a 400 μMstock solution, respectively, and stored at −20° C. until use.

For each template, 60 μl of magnetic 5.91 μM (Bangs Laboratories, Inc.Fishers, Ind.) streptavidin-coated beads, stored as an aqueous, bufferedsuspension (8.57×10⁴ beads/4), at 4° C., were prepared by washing with120 μA bead wash buffer three times and then incubating with templates1, 2, 3 and 4 (T1, T2, T3, T4: Table 1) with biotin on the 5′ end,respectively.

Due to the strong covalent binding affinity of streptavidin for biotin(Kd˜10-15), these magnetic beads are used to immobilize the templates ona solid support, as described below. The reported binding capacity ofthese beads for free biotin is 0.650 μmol/μL of bead stock solution. Fora small (<100 bases) biotinylated ssDNA template, it was conservativelycalculated that 9.1×10⁵ templates could be bound per bead. The beads areeasily concentrated using simple magnets, as with the Dynal MagneticParticle Concentrator or MPC-s (Invitrogen, Carlsbad, Calif.). The MPC-swas used in the described experiments.

An MPC-s was used to concentrate the beads for 1 minute between eachwash, buffer was then added and the beads were resuspended. Followingthe third wash the beads were resuspended in 120 μL bead wash bufferplus 1 μl of each template (40 μM). Beads were incubated for 30 minuteswith rotation (Labquake Tube Rotator, Barnstead, Dubuque, Iowa).Following the incubation, beads were then washed three times in 120Annealing Buffer (20 mM Tris-HCl, 5 mM magnesium acetate, pH 7.5), andre-suspended in 60 μL of the same buffer.

TABLE 1 Sequences for Templates 1, 2, 3, and 4 T1:5′/52Bio/GCA AGT GCC CTT AGG CTT CAG TTC AAA AGT CCT AACTGG GCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 1) T2:5′/52Bio/CCA TGT CCC CTT AAG CCC CCC CCA TTC CCC CCT GAA CCCCCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 2) T3:5′/52Bio/AAG CTC AAA AAC GGT AAA AAA AAG CCA AAA AAC TGGAAA ACA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 3) T4:5′/52Bio/TTC GAG TTT TTG CCA TTT TTT TTC GGT TTT TTG ACC TTTTCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 4)

1.2. Annealing of Sequencing Primer.

The immobilized templates, bound at the 5′ end to 5.91 μm magneticbeads, are then annealed to a 20-base primer complementary to the 3′ endof the templates (Table 1). A 1.0 μL aliquot of the 400 μM primer stocksolution, representing a 20-fold excess of primer to immobilizedtemplate, is then added and then the beads plus template are incubatedwith primer for 15 minutes at 95° C. and the temperature was then slowlylowered to room temperature. The beads were then washed 3 times in 120μL of 25 mM Tricine buffer (25 mM Tricine, 0.4 mg/ml PVP, 0.1% Tween 20,8.8 mM Magnesium Acetate; ph 7.8) as described above using the MPC-s.Beads were resuspended in 25 mM Tricine buffer.

1.3. Incubation of Hybridized Templates/Primer with DNA Polymerase.

Template and primer hybrids are incubated with polymerase essentially asdescribed by Margulies et al. Nature 2005 437(15):376-380 andaccompanying supplemental materials.

2. Loading of Prepared Test Samples onto the ISFET Sensor Array.

The dimensions and density of the ISFET array and the microfluidicspositioned thereon may vary depending on the application. A non-limitingexample is a 512×512 array. Each grid of such an array (of which therewould be 262144) has a single ISFET. Each grid also has a well (or asthey may be interchangeably referred to herein as a “microwell”)positioned above it. The well (or microwell) may have any shapeincluding columnar, conical, square, rectangular, and the like. In oneexemplary conformation, the wells are square wells having dimensions of7×7×10 μm. The center-to-center distance between wells is referred toherein as the “pitch”. The pitch may be any distance although it ispreferably to have shorter pitches in order to accommodate as large ofan array as possible. The pitch may be less than 50 μm, less than 40 μm,less than 30 μm, less than 20 μm, or less than 10 μm. In one embodiment,the pitch is about 9 μm. The entire chamber above the array (withinwhich the wells are situated) may have a volume of equal to or less thanabout 30 μL, equal to or less than about 20 μL, equal to or less thanabout 15 μL, or equal to or less than 10 μL. These volumes thereforecorrespond to the volume of solution within the chamber as well.

2.1 Loading of Beads in an ‘Open’ System.

Beads with templates 1-4 were loaded on the chip (10 μL of eachtemplate). Briefly, an aliquot of each template was added onto the chipusing an Eppendorf pipette. A magnet was then used to pull the beadsinto the wells.

2.2 Loading of Beads in a ‘Closed’ System.

Both the capture beads the packing beads are loaded using flow.Microliter precision of bead solution volume, as well as positioning ofthe bead solution through the fluidics connections, is achieved as shownin FIG. 62 using the bead loading fitting, which includes a majorreservoir (approx. 1 mL in volume), minor reservoir (approx. 10 μL involume), and a microfluidic channel for handling small volumes of beadsolution. This method also leverages the microliter precision of fluidapplication allowed by precision pipettes.

The chip comprising the ISFET array and flow cell is seated in the ZIF(zero insertion force) socket of the loading fixture, then attaching astainless steel capillary to one port of the flow cell and flexiblenylon tubing on the other port. Both materials are microfluidic-typefluid paths (e.g., on the order of <0.01″ inner diameter). The beadloading fitting, consisting of the major and minor reservoirs, itattached to the end of the capillary. A common plastic syringe is filledwith buffer solution, then connected to the free end of the nylontubing. The electrical leads protruding from the bottom of the chip areinserted into a socket on the top of a fixture unit (not shown).

The syringe is pushed to inject the buffer solution through the tubing,across the flow cell (and chip surface) and up thiough the capillary asshown in FIG. 63. This process is called priming, and ensures that thefluidic circuit is free of air. The buffer is injected until the levelof the liquid can be seen at the top of the minor reservoir, through thetransparent major reservoir.

Next, a solution containing the nucleic acid-coated beads is appliedwith a precision pipette to the minor reservoir, as shown in FIG. 64.This application produces a large droplet at the top of the reservoir.The volume of solution added is equal to the volume of the flow chamber(e.g., on the order of 10 μL), and has a concentration of beads, suchthat when added to the volume of buffer solution initially in the minorreservoir, produces the desired concentration of beads to be deliveredto the flow cell. The pipette is retracted, and the syringe is pulledcarefully and slowly until the droplet recedes to the top of the minorreservoir, again viewed through the transparent major reservoir as shownin FIG. 65. Because the microfluidic channel extending down from theminor reservoir is very small (e.g., on the order of 0.01″ diameter),very little mixing occurs between the bead solution and buffer solutionin the fluidic path during this process.

At this point, the bead solution is loaded into the fluidic path, but isnot yet at the location of flow cell. Before transferring the beadsolution plug, or volume of bead solution in the fluidic path, thesolution in the minor reservoir is cleaned. First, approximately 1 mL ofbuffer solution is injected into the major reservoir, effectivelydiluting the bead solution left in the minor reservoir, as shown in FIG.66. Then, the solution is pipetted out by placing a pipette tip alongthe bottom edge of the major reservoir. The level of solution in theminor reservoir is left at its top as shown in FIG. 67.

A volume of buffer solution is then added as droplet above the minorreservoir, as in prior bead solution application as shown in FIG. 68.The volume of this solution is equal to the volume of the fluidic pathbetween the minor reservoir and the flow chamber of the flow cell (i.e.,the microfluidic channel volume plus the capillary volume plus thevolume of flow cell before the flow chamber). Again, the syringe ispulled until the droplet retracts to the top of the minor reservoir asshown in FIG. 69. Now the bead solution plug is loaded into the flowcell's flow chamber.

The loading fixture is now lifted and placed over a pyramidal base,containing a magnet at its apex, as shown in FIG. 70. The magnet pullsthe beads from the bead solution into the microwells of the chip. Thefixture is removed from the base after a few seconds. The entireprocess, excluding the initial priming of the fluidics with buffersolution, can be repeated for the loading of small packing beads intothe microwells, if necessary.

It will be appreciated that there will be other ways of drawing thebeads into the wells of the flow chamber, including centrifugation orgravity. The invention is not limited in this respect.

3. Dna Sequencing Using the ISFET Sensor Array.

3.1 DNA Sequencing in an ‘Open’ System.

The results shown are representative of an experiment carried out in an‘open’ system (i.e., the ISFET chip is placed on the platform of theISFET apparatus and then each nucleotide (5 μL resulting in 6.5 each)was manually added in the following order: dATP, dCTP, dGTP and dTTP(100 mM stock solutions, Pierce, Milwaukee, Wis.), by pipetting thegiven nucleotide into the liquid already on the surface of the chip andcollecting data from the chip at a rate of 2.5 mHz. This resulted indata collection over 7.5 seconds at approximately 18 frames/second. Datawere then analyzed using LabView.

Given the sequences of the templates, addition of dATP resulted in a 4base extension in template 4. Addition of dCTP resulted in a 4 baseextension in template 1. Addition of dGTP caused template 1, 2 and 4 toextend as indicated in Table 2 and addition of dTTP results in a run-off(extension of all templates as indicated).

FIG. 71 (A-D) illustrates the extension reactions. In the left panel,all pixels for one snapshot in time are shown, and on the right, mV vs.time graphs are shown for four selected pixels from the set on the left.White arrows indicate active pixels where extensions are taking place.In the run-off (FIG. 71D) in addition to the wells marked in FIG. 71C,an additional arrow indicates a well where an extension did not takeplace following the addition of dGTP, but rather dATP and is then seenagain during the run-off.

Preferably when the method is performed in a non-automated manner (i.e.,in the absence of automated flow and reagent introduction), each wellcontains apyrase in order to degrade the unincorporated dNTPs. It is tobe understood that apyrase can be substituted, in this embodiment or inany other embodiment discussed herein, with another compound (or enzyme)capable of degrading dNTPs.

TABLE 2 Set-up of experiment and order of nucleotide addition. dATP dCTPdGTP dTTP T1 0 (3:C; 1 Run-off (25) 1:A) 4 T2 0 0 4 Run-off (26) T3 0 00 Run-off (30) T4 4 0 2 Run-off (24)

3.2 DNA Sequencing Using Microfluidics on Sensor Chip

Sequencing in the flow regime is an extension of open application ofnucleotide reagents for incorporation into DNA. Rather than add thereagents into a bulk solution on the ISFET chip, the reagents are flowedin a sequential manner across the chip surface, extending a single DNAbase(s) at a time. The dNTPs are flowed sequentially, beginning withdTTP, then dATP, dCTP, and dGTP. Due to the laminar flow nature of thefluid movement over the chip, diffusion of the nucleotide into themicrowells and finally around the nucleic acid loaded bead is the mainmechanism for delivery. The flow regime also ensures that the vastmajority of nucleotide solution is washed away between applications.This involves rinsing the chip with buffer solution and apyrase solutionfollowing every nucleotide flow. The nucleotides and wash solutions arestored in chemical bottles in the system, and are flowed over the chipusing a system of fluidic tubing and automated valves. The ISFET chip isactivated for sensing chemical products of the DNA extension duringnucleotide flow.

The invention claimed is:
 1. A method of operating an apparatuscomprising an array of chemically sensitive sensors arranged in rows andcolumns, comprising: producing signal samples in a plurality of sensorsof the array in response to a first timing signal; enabling an outputcircuit coupled to the plurality of sensors in response to a selectsignal; outputting the signal samples to the output circuit from theplurality of sensors; and applying a reference voltage to a referencecell, and outputting a reference signal to the output circuit from thereference cell.
 2. The method of claim 1, including applying a chemicalsample and a reference electrode to chemical sample wells coupled to theplurality of sensors.
 3. The method of claim 1, wherein each sensor inthe array comprises a chemically-sensitive field effect transistor(chemFET) including a floating gate and a passivation layer, and thesignal samples indicate an ion-concentration in an analyte solutioncoupled to the corresponding floating gates via the passivation layers.4. The method of claim 1, including performing a calibration process todetermine the reference voltage.
 5. The method of claim 1, wherein theplurality of sensors comprises a column of chemically-sensitive fieldeffect transistors (chemFETs), each having a floating gate structurecoupled to a corresponding chemical sample well, and wherein thereference cell comprises an extra chemFET in the column having afloating gate structure tied to a reference voltage connector.
 6. Themethod of claim 1, wherein the plurality of sensors comprises a row ofchemically-sensitive field effect transistors (chemFETs), each having afloating gate structure coupled to a corresponding chemical sample well,and wherein the reference cell comprises an extra chemFET in the rowhaving a floating gate structure tied to a reference voltage connector.7. The method of claim 1, further comprising: disposing a plurality oftemplate nucleic acids in chemical sample wells for the plurality ofsensors of the array; and synthesizing a new nucleic acid strand from atleast one template nucleic acid disposed in the chemical sample wells byincorporating one or more known nucleotides to the at least one templatenucleic acid, and wherein the signal samples produced by the pluralityof sensors indicate incorporation of the one or more known nucleotides.8. The method of claim 7, further comprising using the signal samplesproduced by the plurality of sensors to determine at least a portion ofa sequence of the new nucleic acid strand.
 9. The method of claim 8,further comprising using the reference signal from the reference cell todetermine the portion of the sequence.
 10. A method of operating anapparatus comprising an array of cells comprising field effecttransistors (FETs), the method comprising: operating FETs of cells in areference portion of the array as dummy sensors to produce referencesignals; operating FETs of cells in an active portion of the arraycoupled to corresponding chemical sample wells to produce signalsamples; and using the reference signals from the FETs of cells in thereference portion to calibrate operation of the FETs of cells in theactive portion of the array.
 11. The method of claim 10, wherein theFETs of cells in the reference portion and in the active portion includefloating gate structures, and operating FETs of the cells in thereference portion of the array includes applying a reference voltage totheir floating gate structures.
 12. The method of claim 10, wherein thearray is arranged in rows and columns, and wherein the reference portionincludes a column of cells in the array.
 13. The method of claim 10,wherein the array is arranged in rows and columns, and wherein thereference portion includes a column of cells in the array, and operatingthe cells in the active portion and in the reference portion of thearray includes selectively connecting the cells in corresponding columnsof the array to column output circuits, including a column outputcircuit selectively connectable to the cells in the column in thereference portion.
 14. The method of claim 10, wherein the array isarranged in rows and columns, and wherein the reference portion includesa row of cells in the array.
 15. The method of claim 10, wherein thearray is arranged in rows and columns, and wherein the reference portionincludes a row of cells in the array, and operating the cells in theactive portion and in the reference portion of the array includesselectively connecting the cells in rows of the array to correspondingcolumn output circuits, including a cell in the row in the referenceportion.
 16. The method of claim 10, further comprising: disposing aplurality of template nucleic acids in chemical sample wells for FETs inthe active portion of the array; and synthesizing a new nucleic acidstrand from at least one template nucleic acid disposed in the chemicalsample wells by incorporating one or more known nucleotides to the atleast one template nucleic acid, and wherein the signal samples producedby the cells in the active region indicate incorporation of the one ormore known nucleotides.
 17. The method of claim 16, further comprisingusing the signal samples produced by the cells in the active portion todetermine at least a portion of a sequence of the new nucleic acidstrand.
 18. The method of claim 16, further comprising using thereference signals from the cells in the reference portion to determineat least a portion of a sequence of the new nucleic acid strand.
 19. Themethod of claim 10, including selecting a cell in a row in the arrayusing a column select signal.
 20. The method of claim 10, includingactivating cells in the array using a row select signal.
 21. The methodof claim 10, wherein operating the cells in the active portion and inthe reference portion of the array includes connecting selected cells toa sample and hold capacitor using a column select switch.
 22. The methodof claim 10, wherein including applying a reference voltage to ananalyte solution in the chemical sample wells of the cells in the activeportion.
 23. The method of claim 10, wherein the FETs in the cells inthe active portion have thresholds that indicate an ion-concentration inan analyte solution in the chemical sample wells.